Caspase-8 binding protein, its preparation and use

ABSTRACT

The present invention relates to a caspase-8 interacting polypeptide (Cari), methods for its preparation, and its use.

FIELD OF THE INVENTION

The present invention relates to a caspase-8 interacting polypeptide(Cari), methods for its preparation, and its use.

BACKGROUND OF THE INVENTION

Tumor Necrosis Factor (TNF-alpha) and Lymphotoxin (TNF-beta) aremultifunctional pro-inflammatory cytokines formed mainly by mononuclearleukocytes, which have many effects on cells (Wallach D. (1986); andBeutler and Cerami (1987)). Both TNF-alpha and TNF-beta initiate theireffects by binding to specific cell surface receptors. Some of theeffects are likely to be beneficial to the organism: they may destroy,for example, tumor cells or virus infected cells and augmentantibacterial activities of granulocytes. In this way, TNF contributesto the defense of the organism against tumors and infectious agents andcontributes to the recovery from injury. Thus, TNF can be used as ananti-tumor agent in which application it binds to its receptors on thesurface of tumor cells and thereby initiates the events leading to thedeath of the tumor cells. TNF can also be used as an anti-infectiousagent.

However TNF-alpha has deleterious effects. There is evidence thatoverproduction of TNF-alpha may play a major pathogenic role in severaldiseases. For example, effects of TNF-alpha, primarily on thevasculature, are known to be a major cause for symptoms of septic shock(Tracey et al, 1994). 1994). In some diseases, TNF may cause excessiveloss of weight (cachexia) by suppressing activities of adipocytes and bycausing anorexia, and TNF-alpha was thus called cachectin. It was alsodescribed as a mediator of the damage to tissues in rheumatic diseases(Beutler and Cerami, 1987) and as a major mediator of the damageobserved in graft-versus-host reactions (Grau et al, 1989). In addition,TNF is known to be involved in the process of inflammation and in manyother diseases.

Two distinct, independently expressed receptors, the p55 (CD120a) andthe p75 (CD120b) TNF-receptors, which bind both TNF-alpha and TNF-betaspecifically, initiate and/or mediate the above noted biological effectsof TNF. These two receptors have structurally dissimilar intracellulardomains suggesting that they signal differently (See Hohmann et al,1989; Engelmann et al, 1990a and b; Brockhaus et al, 1990; Loetscher etal, 1990; Schall et al, 1990; Nophar et al, 1990; Smith et al, 1990).However, the cellular mechanisms, for example, the various proteins andpossibly other factors, which are involved in the intracellularsignaling of the CD120a and CD120b, have yet to be elucidated. It isintracellular signaling, which occurs usually after the binding of theligand, i.e., TNF (alpha or beta), to the receptor that is responsiblefor the commencement of the cascade of reactions that ultimately resultin the observed response of the cell to TNF.

As regards the above-mentioned cytocidal effect of TNF, in most cellsstudied so far, this effect is triggered mainly by CD120a. Antibodiesagainst the extracellular domain (ligand binding domain) of CD120a canthemselves trigger the cytocidal effect (see EP 412486) which correlateswith the effectiveness of receptor cross-linking by the antibodies,believed to be the first step in the generation of the intracellularsignaling process. Further, mutational studies (Brakebusch et al, 1992;Tartaglia et al, 1993) have shown that the biological function of CD120adepends on the integrity of its intracellular domain, and accordingly ithas been suggested that the initiation of intracellular signalingleading to the cytocidal effect of TNF occurs as a consequence of theassociation of two or more intracellular domains of CD120a. Moreover,TNF (alpha and beta) occurs as a homotrimer, and as such, has beensuggested to induce intracellular signaling via CD120a by way of itsability to bind to and to cross-link the receptor molecules, i.e., causereceptor aggregation (Engelmann et al 1990b).

Another member of the TNF/NGF superfamily of receptors is the FAS/APO1receptor (CD95). CD95 mediates cell death in the form of apoptosis (Itohet al, 1991), and appears to serve as a negative selector ofautoreactive T cells, i.e., during maturation of T cells, CD95 mediatesthe apoptotic death of T cells recognizing self-antigens. It has alsobeen found that mutations in the CD95 gene (lpr) cause alymphoproliferation disorder in mice that resembles the humancell-surface associated molecule carried by, amongst others, killer Tcells (or cytotoxic T lymphocytes—CTLs), and hence when such CTLscontact cells carrying CD95, they are capable of inducing apoptotic celldeath of the CD95-carrying cells. Further, monoclonal antibodies havebeen prepared that are specific for CD95, these monoclonal antibodybeing capable of inducing apoptotic cell death in cells carrying CD95,including mouse cells transformed by cDNA encoding human CD95 (e.g.,Itoh et al, 1991).

TNF receptor and Fas signaling mechanisms comprising the differentreceptors, their regulation, and the down stream signaling moleculesidentified are reviewed in detailed by Wallach et al (1999).

It has been found that certain malignant cells and HIV-infected cellscarry CD95 on their surface, antibodies against CD95, or the CD95ligand, may be used to trigger the CD95 mediated cytotoxic effects inthese cells and thereby provide a means for combating such malignantcells or HIV-infected cells (see Itoh et al, 1991). Finding yet otherways for enhancing the cytotoxic activity of CD95 may therefore alsohave therapeutic potential.

It has been a long felt need to provide a way for modulating thecellular response to TNF (alpha or beta) and CD95 ligand. For example,in the pathological situations mentioned above, where TNF or CD95 ligandis overexpressed, it is desirable to inhibit the TNF- or CD95ligand-induced cytocidal effects, while in other situations, e.g., woundis desirable to inhibit the TNF- or CD95 ligand-induced cytocidaleffects, while in other situations, e.g., wound healing applications, itis desirable to enhance the TNF effect, or in the case of CD95, in tumorcells or HIV-infected cells, it is desirable to enhance the CD95mediated effect.

A number of approaches have been made by the applicants (see, forexample, European patent specifications of EP 186,833, EP 308,378, EP398,327 and EP 412,486) to regulate the deleterious effects of TNF byinhibiting the binding of TNF to its receptors using anti-TNF antibodiesor by using soluble TNF receptors (being essentially the solubleextracellular domains of the receptors) to compete with the binding ofTNF to the cell surface-bound TNF-receptors (TNF-Rs). Further, on thebasis that TNF-binding to its receptors is required for the TNF-inducedcellular effects, approaches by applicants (see, for example, EP568,925) have been made to modulate the TNF effect by modulating theactivity of the TNF-Rs.

EP 568,925 relates to a method of modulating signal transduction and/orcleavage in TNF-Rs whereby peptides or other molecules may interacteither with the receptor itself or with effector proteins interactingwith the receptor, thus modulating the normal function of the TNF-Rs. InEP 568,925, there is described the construction and characterization ofvarious mutant forms of CD120a, having mutations in its extracellular,transmembrane and intracellular domains. In this way, regions within theabove domains of CD120a were identified as being essential to thefunctioning of the receptor, i.e., the binding of the ligand (TNF) andthe subsequent signal transduction and intracellular signaling whichultimately results in the observed TNF-effect on the cells. Further,there are also described a number of approaches to isolate and identifyproteins, peptides or other factors which are capable of binding to thevarious regions in the above domains of CD120a, which proteins, peptidesand other factors may be involved in regulating or modulating theactivity of TNF-Rs. A number of approaches for isolating and cloning theDNA sequences encoding such proteins and peptides; for constructingexpression vectors for the production of these proteins and peptides;and for the preparation of antibodies or fragments thereof whichinteract with CD120a or with the above proteins and peptides that bindvarious regions of CD120a, are also set forth in EPO 368,925. However,EP 568,925 does not specify the actual proteins and peptides that bindto the intracellular domains of the TNF-Rs. Similarly, in EP 568,925there is no disclosure of specific proteins or peptides capable ofbinding the intracellular domain of CD95.

Thus, when it is desired to inhibit the effect of TNF, or of the CD95ligand, it would be desirable to decrease the amount or the activity ofTNF-Rs or CD95 at the cell surface, while an increase in the amount orthe activity of TNF-R or CD95 would be desired when an enhanced TNF orCD95 ligand effect is sought. To this end the promoters of both theCD120a and the CD120b have been sequenced, analyzed and a number of keysequence motifs have been found that are specific to varioustranscription regulating factors, and as such the expression of theseTNF-Rs can be controlled at their promoter level, i.e., inhibition oftranscription from the promoters for a decrease in the number ofreceptors, and an enhancement of transcription from the promoters for anincrease in the number of receptors (EP 606,869 and WO 95/31206).

While it is known that the tumor necrosis factor (TNF) receptors, andthe structurally related receptor CD95, trigger in cells, uponstimulation by leukocyte-produced ligands, destructive activities thatlead to their own demise, the mechanisms of this triggering are stilllittle understood. Mutational studies indicate that in CD95 and CD120asignaling for cytotoxicity involve distinct regions within theirintracellular domains (Brakebusch et al, 1992; Tartaglia et al, 1993;Itoh and Nagata, 1993). These regions (the ‘death domains’) havesequence similarity. The ‘death domains’ of both CD95 and CD120a tend toself-associate. Their self-association apparently promotes the receptoraggregation, which is necessary for initiation of signaling (see Bigdaet al, 1994; Boldin et al, 1995), and at high levels of receptorexpression can result in triggering of ligand-independent signaling(Boldin et al, 1995).

Some of the cytotoxic effects of lymphocytes are mediated by interactionof a lymphocyte-produced ligand with CD95 in target cells (see alsoNagata and Goldstein, 1995). Cell killing by mononuclear phagocytesinvolves TNF and its receptor CD120a (see also Vandenabeele et al,1995). Like other receptor-induced effects, cell death induction by theTNF receptors and CD95 occurs via a series of protein-proteininteractions, leading from ligand-receptor binding to the eventualactivation of enzymatic effector functions, which have been shown tocomprise non-enzymatic protein-protein interactions that initiatesignaling for cell death: binding of trimeric TNF or the CD95 ligandmolecules to the receptors, the resulting interactions of theirintracellular domains (Brakebusch et al, 1992; Tartaglia et al, 1993;Itoh and Nagata, 1993) augmented by a propensity of the death-domainmotifs to self-associate (Boldin et al, 1995a), and induced binding oftwo cytoplasmic proteins (which can also bind to each other) to thereceptors' intracellular domains—MORT-1 (or FADD) to CD95 (Boldin et al,1995b; Chinnaiyan et al, 1995; Kischkel et a, 1995) and TRADD to CD120a(Hsu et al, 1996). Besides their binding to CD95 and CD120a, MORT-1 andTRADD are also capable of binding to each other, as well as to otherdeath domain containing proteins, such as RIP (Stanger et al, 1995),which provides for a functional “cross-talk” between CD95 and CD120a.These bindings occur through a conserved sequence motif, the ‘deathdomain module’ common to the receptors and their associated proteins.Furthermore, although in the yeast two-hybrid test MORT-1 was shown tobind spontaneously to CD95, in mammalian cells, this binding takes placeonly after stimulation of the receptor, suggesting that MORT-1participates in the initiating events of CD95 signaling. MORT-1 does notcontain any sequence motif characteristic of enzymatic activity, andtherefore, its ability to trigger cell death seems not to involve anintrinsic activity of MORT-1 itself, but rather, activation of someother protein(s) that bind MORT-1 and act further downstream in thesignaling cascade. Cellular expression of MORT-1 mutants lacking theN-terminal part of the molecule have been shown to block cytotoxicityinduction by CD95 or CD120a (Hsu et al, 1996; Chinnaiyan et al, 1996),indicating that this N-terminal region transmits the signaling for thecytocidal effect of both receptors through protein-protein interactions.

Recent studies have implicated a group of cytoplasmic thiolproteases,which are structurally related to the Caenorhabditis elegans proteaseCED3 and to the mammalian interleukin-1 beta-converting enzyme (ICE) inthe onset of various physiological cell death processes (reviewed inKumar, 1995 and Henkart, 1996). There is also evidence that protease(s)of this family take part in the cell-cytotoxicity induced by CD95 andTNF-Rs. Specific peptide inhibitors of the proteases and twovirus-encoded proteins that block their function, the cowpox proteinCrmA and the Baculovirus p35 protein, were found to provide protectionto cells against this cell-cytotoxicity (Enari et al, 1995; Tewari etal, 1995; Xue et al, 1995; Beidler et al, 1995). Rapid cleavage ofcertain specific cellular proteins, apparently mediated by protease(s)of the CED3/ICE (caspase) family, could be demonstrated in cells shortlyafter stimulation of CD95 or TNF-Rs.

One such protease and various isoforms thereof (including inhibitoryones), is known as MACH (now caspase-8) which is a MORT-1 bindingprotein has been isolated, cloned, characterized, and its possible usesalso described, as is set forth in detail and incorporated herein intheir entirety by reference, in co-owned PCT/US96/10521, and in apublication of the present inventors (Boldin et al, 1996). Another suchprotease and various isoforms thereof (including inhibitory ones),designated Mch4 (also called caspase-10) has also been isolated andcharacterized by the present inventors (unpublished) and others(Fernandes-Alnemri et al, 1996; Srinivasula et al, 1996). Caspase-10 isalso a MORT-1 binding protein. Thus, details concerning all aspects,features, characteristics and uses of caspase-10 are set forth in theabove noted publications, all of which are incorporated herein in theirentirety by reference.

It should also be noted that the caspases, caspase-8 and caspase-10,which have similar pro-domains (see Boldin et al, 1996; Muzio et al,1996; Fernandes-Alnemri et al, 1996; Vincenz and Dixit, 1997) interactthrough their pro-domains with MORT-1, this interaction being via the‘death effector domain’, DED, present in the N-terminal part of MORT-1and present in duplicate in caspase-8 and caspase-10 (see Boldin et al,1995b; Chinnaiyan et al, 1995).

The caspases (cysteine aspartate-specific proteinases) are a growingfamily of cysteine proteases that share several common features. Most ofthe caspases have been found to participate in the initiation andexecution of programmed cell death or apoptosis, while the others appearto be involved in the production of proinflammatory cytokines (Nicholsonand Thornberry et al, 1997, Salvesen et al, 1997, Cohen, 1997). They aresynthesized as catalytically almost inactive precursors and aregenerally activated by cleavage after specific internal aspartateresidues present in interdomain linkers. The cleavage sites of caspasesare defined by tetrapeptide sequences (X-X-X-D) and cleavage alwaysoccurs downstream of the aspartic acid. As a result certain matureactive caspases can process and activate their own as well as otherinactive precursors (Fernandes-Alnemri et al, 1996, Srinivasula et al,1996).

Activation of the programmed cell death process is generally specificand involves sequential processing of downstream caspases named“executioner” caspases by upstream caspases named “initiator” caspases.The functional characteristics of the two classes of caspases are alsoreflected by their structure. In fact the “initiator caspases” containlonger pro-domain regions as compared to the “executioner” caspases(Salvesen et al, 1997; Cohen, 1997). The long pro-domain allows theinitiator or “‘apical” caspases to be activated by triggering of thedeath receptors of the TNF receptor family. Upon ligand-inducedtrimerization of the death receptors, the initiator caspases arerecruited through their long N-terminal pro-domain to interact withspecific adapter molecules to form the death inducing signaling complex(Cohen, 1997; Kischkel et al, 1995). For example, caspase-8/MACH andprobably caspase-10, which contain two DEDs, are recruited to thereceptor complex by the adapter molecules FADD/MORT-1, whereas caspase-2is assumed to be recruited by CRADD/RAIDD and RIP (Nagata et al, 1997;MacFarlane et al, 1997; Ahmad et al, 1997; Duan and Dixit, 1997). Due tothe trimeric nature of the activated receptor complex, at least twocaspase molecules are thought to be brought in close proximity to eachother, thus leading to their activation by auto-catalytic processing(Yang et al, 1998; Muzio et al, 1998).

Caspases are synthesized as pro-enzymes consisting of three majorsubunits, the N-terminal pro-domain, and two subunits, which aresometimes separated by a linker peptide. The two subunits have beentermed “long” or subunit 1 (Sub-1) containing the major part of theactive enzymatic site, and “short” or subunit 2 (Sub-2). For fullactivation of the enzyme, it is processed to form the pro-domain and thetwo sub-domains. The two subunits form a heterodimer. Based on thededuced three dimensional structure of caspase-3, it appears that theC-terminal end of the long domain as well as the N-terminus of the shortsub-domain have to be freed and the C-terminus of the short subunit hasto be brought into close proximity with the N-terminus of the longsubunit in order to yield a correctly folded and active enzyme (Rotondaet al, 1996; Mittl et al, 1997; Srinivasula et al, 1998).

Although pathways leading to apoptosis or necrosis have always beenconsidered to be completely distinct, recent findings have suggestedthat the caspases, which represent the main mediators of apoptosis, canalso be implicated in necrosis both in a negative and a positive manner.Indeed, overexpression of the caspase inhibitor CrmA in L929 cells wasshown to increase by a factor of 1000 the sensitivity of these cells forthe necrotic activity of TNF (Vercammen et al, 1998), indicating aninhibitory role of caspases on TNF-induced necrotic activity. Moreover,the TNFR1- and Fas-associated death domains that play a crucial role inapoptosis induction by these ligands (reviewed in Wallach et al, 1999),were recently also suggested to play an important role in necrosisinduction (Boone et al, 2000). Interestingly, the FasL-induced livernecrosis was shown to be blocked by caspase inhibitors (Kunstleet al,1997).

Because caspase-mediated proteolysis is critical and central element ofthe apoptotic process (Nicholson and Thornberry, 1997; Villa et al,1997; and Salvesen and et al, 1997), identification of the crucialdownstream molecular targets of these proteases is inevitable forunderstanding apoptotic signal transduction. Various structural andsignaling proteins have been shown to be cleaved by caspases duringapoptotic death (Nicholson and Thornberry, 1997; Villa. et al., 1997)including ICAD, an inhibitor of caspase-activated Dnase, which isessential for internucleosomal DNA degradation but not for execution ofapoptosis (Enari et al, 1998; Sakahira et al, 1998). Gelsolin, anactin-regulatory protein that modulates cytoplasmic actin gelsoltransformation (Yin and Stossel, 1979), is implicated in apoptosis onthe basis of (i) its cleavage during apoptosis in vivo (Kothakota et al,1997) (ii) prevention of apoptosis by its overexpression (Ohtsu et al,1997) and (iii) induction of apoptosis by one of the cleaved productsKothakota et al, 1997). Gelsolin has Ca+2 activated multiple activities,severs actin filaments, and caps the fast growing ends of filaments, andalso nucleates actin polymerization (Yin and Stossel, 1980; Kurth andBryan, 1984; Janmey and Stossel, 1987).

Application WO 00/39160 discloses caspase-8 interacting proteins capableof interacting with Sub-1 and/or Sub-2 of caspase-8. The caspaseinteracting proteins were discovered by two-hybrid screen using singlechain construct of caspase-8.

Application WO 98/30582 (Jacobs et al) discloses nucleotide and thepredicted amino acid sequences of secreted or membrane proteinDF518_(—)3 isolated from a human adult brain cDNA library. The proteinwas identified by using methods that are selective for cDNAs encodingsecreted proteins (U.S. Pat. No. 5,536,637), and was also identified asencoding a secreted or transmembrane protein on the basis of computeranalysis of the amino acid sequence of the encoded protein. The proteinaccording to the present invention differs from DF518_(—)3 in itslocation (intracellular versus membrane/secreted) and its amino acidsequence (has one non-conservative amino acid change in residue 230 Eversus G). In the WO application numerous non-related activities thatare not supported by any data, are attributed to DF518_(—)3.

SUMMARY OF THE INVENTION

The present invention relates to an intracellular polypeptide (Cari)capable of interacting with a pro-caspase or mutein or fragment thereof,which polypeptide comprises the amino acid sequence of SEQ ID NO:3, oran isoform, a mutein except DF518_(—)3, an allelic variant, fragment,fusion protein, or derivative thereof. In one embodiment, thepolypeptide of the invention is cleavable in vitro and in vivo bycaspases, preferably caspase-8.

In addition, the invention provides for a Cari polypeptide mutein havinga dominant-negative effect on the activity of the endogenous Caripolypeptide and muteins capable of inhibiting or increasing thecytotoxic effect of a caspase, more preferably, caspase-8.

In one embodiment, the invention provides a non-cleavable Cari mutant(Cari D600E) polypeptide, wherein the amino acid residue in whichresidue D600 in the Cari polypeptide is replaced with the glutamic acidresidue. This polypeptide is capable of increasing the cytotoxic effectof caspase-8. In another embodiment, the invention provides peptidesderived from Cari responsible for binding caspase-8 such as the onescomprising the amino acid sequences in SEQ ID NO:4 and SEQ ID NO:5.

Furthermore, the present invention provides a DNA sequence encoding aCari polypeptide, or an isoform, allelic variant, fragment, mutein(e.g., Cari D600E), fusion protein, or derivative thereof, a DNAsequence capable of hybridizing under moderately stringent conditions toa DNA sequence encoding a Cari polypeptide, or an isoform, allelicvariant, fragment, mutein, fusion protein, or derivative thereof, or toa DNA sequence corresponding to SEQ ID NO:2.

More specifically the present invention provides a DNA sequence encodingthe polypeptide of SEQ ID NO:3. The invention also provides the DNA of anon-cleavable Cari mutant (e.g., Cari D600E), and the DNA encodingpeptides (e.g., SEQ ID NO:4, SEQ ID NO:5). In addition, the presentinvention provides also a ribozyme and an antisense oligonucleotidecomprising at least 9 nucleotides corresponding to the above DNAsequence, preferably the antisense oligonucleotide of SEQ ID NO:6 andSEQ ID NO:7.

The present invention also provides vectors comprising the DNA sequenceencoding a Cari polypeptide, or its isoform, allelic variant, fragment,mutein, fusion protein, or derivative thereof, and methods for theproduction of the Cari polypeptide, or its isoform, allelic variant,fragment, mutein, fusion protein, or derivative thereof by introducingsaid vector in prokaryotic or eukaryotic host cells, preferably, amammalian, insect, or yeast cell, and more preferably in cells selectedfrom HeLa, 293 T HEK and CHO cells and growing the cells and isolatingthe protein produced.

Moreover, the invention provides a viral vector encoding a Caripolypeptide, or its isoform, allelic variant, fragment, mutein, a fusionprotein, a ribozyme, an antisense oligonucleotide or derivative thereofand its use for introducing into mammalian cells a Cari polypeptide,isoform, allelic variant, fragment, mutein, fusion protein.

In addition the invention provides a vector suitable for targetingregulatory sequences functional in cells for the activation of theendogenous Cari or an inhibitor of Cari expression.

In another aspect the invention provides for a polyclonal or monoclonalantibody, chimeric antibody, fully humanized antibody, anti-anti-Idantibody or fragment thereof directed at an epitope of a Caripolypeptide, or its isoform, allelic variant, fragment, mutein, fusionprotein, or derivative thereof and its use for diagnostic purposes ordevelopment in immunoassays for the detection of a Cari polypeptide, orits isoform, allelic variant, fragment, mutein, a fusion protein, orderivative thereof in biological fluids.

Furthermore, the invention provides a host cell selected fromprokaryotic or eukaryotic cells, preferably HeLa, 293 T HEK and CHOcells, comprising a vector encoding Cari, and a method of producing Carior an isoform, mutein, allelic variant, fragment, fusion protein orderivative thereof. Alternatively, the invention provides a method ofproducing Cari or an isoform, mutein, allelic variant, fragment, fusionprotein or derivative thereof comprising the generation of a transgenicanimal and isolating the protein produced from the body fluids of theanimal.

In one aspect, the invention provides a method of gene therapy fortreatment of an inflammatory disease selected from multiple sclerosiswith primary oligodendrogliopathy, autoimmune uveoretinitis, diabetes,lupus, autoimmune myocarditis I, HCV mediated chronic hepatitis, chronicgastritis, e.g., type A gastritis, mixed connective tissue disease(MCTD), Crohn's disease, or ulcerative colitis, comprising inducing theexpression of Cari or a mutein, (e.g., Cari D600E), fragment (e.g., SEQID NO:4, SEQ ID NO:5), antisense preferably of SEQ ID NO-6 and/or SEQ IDNO:7 and a ribozyme of Cari at a desired site in a human patient inneed.

In addition, the invention provides a method of treatment andinflammatory disease selected from multiple sclerosis with primaryoligodendrogliopathy, autoimmune uveoretinitis, diabetes, lupus,autoimmune myocarditis I, HCV mediated chronic hepatitis, chronicgastritis, e.g., type A gastritis, mixed connective tissue disease(MCTD), Crohn's disease, or ulcerative colitis, comprising regulation ofendogenous Cari or Cari inhibitor, by targeting a vector having DNAregulatory sequences functional in cells for enabling endogenous geneactivation of Cari or an endogenous Cari inhibitor at a desired site ina human patient in need.

Moreover, the invention provides the use of Cari pqlypeptide, mutein,isoform, allelic variant, fragment (e.g., SEQ ID NO:4, SEQ ID NO:5),fusion protein or derivative thereof, an antisense (e.g., SEQ ID NO:6and/or SEQ ID NO:7), a vector encoding Cari and its fragments, andantibodies against Cari for down-regulation of a caspase, in situationswhere excessive cell death by apoptosis occurs, for example, byinduction of the TNF receptor signaling pathway.

The invention provides further the use of Cari polypeptide, or a mutein(e.g., Cari D600E), an isoform, allelic variant, or fragment, fusionprotein or derivative thereof, a vector encoding Cari and its fragments,a DNA encoding Cari or fragments and muteins, a vector comprising theDNA encoding Cari or fragment or muteins, vectors for endogenous Cariactivation and anti-idiotype antibodies of Cari for up-regulation of acaspase activity and increase of apoptosis in situations where excessivecell death is required.

In another aspect, the invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of Cari polypeptide, or amutein (e.g., Cari D600E), isoform, allelic variant, fragment (e.g. SEQID NO:4, SEQ ID NO:5) fusion protein, or derivative thereof, a DNA orvector encoding Cari or muteins or fragments, a vector for endogenousactivation of Cari or its inhibitor, antisense, ribozyme or an antibodyspecific for Cari, for the treatment of an inflammatory disease selectedfrom multiple sclerosis with primary oligodendrogliopathy, autoimmuneuveoretinitis, diabetes, lupus, autoimmune myocarditis I, HCV mediatedchronic hepatitis, chronic gastritis, e.g., type A gastritis, mixedconnective tissue disease (MCTD), Crohn's disease, or ulcerativecolitis.

In addition, the invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of Cari polypeptide, or amutein (e.g., Cari D600E), isoform, allelic variant, fragment fusionprotein, or derivative thereof, a DNA or vector encoding Cari or muteinsor fragments, a vector for endogenous activation of Cari, or an antiidiotype antibody specific for Cari, for the treatment of cancer.

In another embodiment, the invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of aninhibitor of Cari for the treatment or prevention of a disease in whichthe activity of Cari is involved.

The invention further provides a method for the isolation,identification and cloning of another polypeptide of the same class ofCari comprising the use of a DNA encoding Cari or muteins or fragmentsthereof to screen a DNA library, or a caspase specific antibody suitablefor co-immunoprecipitation of a caspase and bound polypeptide or byaffinity purification of such polypeptides from samples selected frombody fluids, cell extracts and DNA expression libraries with Carispecific antibodies, or by using Cari polypeptide or a mutein, isoform,allelic variant, fragment, fusion protein or derivative thereof as theprey or the bait in the yeast two-hybrid procedure.

The invention also relates to a method for isolating a polypeptide orfactor involved in intracellular signaling processes, from samplesselected from cell extracts human fluids and expression libraries,comprising co-immunoprecipitating Cari and the polypeptides or factorsinvolved in intracellular signaling using an antibody recognizing Cari.

Furthermore, the invention provides a method for screening for a peptideor a small molecule antagonist to Cari, comprising high through putscreening and selection of such molecules able to inhibit theinteraction of Cari to pro-caspase-8 or a mutein (e.g., Cari D600E),isoform, allelic variant, fragment (e.g., SEQ ID NO:4 or SEQ ID NO:5),fusion protein or derivative thereof or selection of molecules able toinhibit apoptosis enhanced by Cari or a mutein, isoform, allelicvariant, fragment, fusion protein or derivative thereof.

In addition, the invention relates to a method of treatment and/orprevention of a disorder selected from, multiple sclerosis with primaryoligodendrogliopathy, autoimmune uveoretinitis, diabetes, lupus,autoimmune myocarditis I, HCV mediated chronic hepatitis, chronicgastritis, e.g., type A gastritis, mixed connective tissue disease(MCTD), Crohn's disease, ulcerative colitis and cancer, comprisingadministering to a patient in need thereof a pharmaceutically effectiveamount of a Cari polypeptide or a mutein (e.g., Cari D600E), isoform,allelic variant, fragment (e.g., SEQ ID NO:4, SEQ ID NO:5), fusionprotein or derivative thereof or a DNA or vectors encoding Cari ormutein or fragments thereof, or vectors for endogenous gene activationof Cari or Cari inhibitor, or a specific antibody for Cari.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the amino acid sequence of pro-caspase-8 (SEQ ID NO:8). Thepeptide sequences from caspase-8 used for the preparation of mAbs are inbold and underlined.

-   -   Peptide 179—The peptide CQGDNYQKGIPVETD (residues 360-374 of SEQ        ID NO:8) corresponding to the C-terminus of the large subunit of        caspase-8 (Sub-1).    -   Peptide 182—The peptide LSSPQTRYIPDEAD (residues 385-398 of SEQ        ID NO:8) corresponding to the N-terminus of the small subunit of        the caspase-8 (Sub-2, residues Leu385-Asp398).    -   Peptide 183—The peptide SESQTLDKVYQMKSKPR (residues 217-233 of        SEQ ID NO:8) corresponding to the N-terminus of Sub-1 (residues        Ser217-Arg233).

FIG. 2 shows the effective immunoprecipitation of minute amounts ofcaspase-8 found in lysates of BJAB cells using a monoclonal antibodyagainst epitope 179. Depletion of caspase-8 from the BJAB cell lysates(prepared before, −, and after, +, Fas receptor stimulation) byimmunoprecipitation with various antibodies is shown from left to right:

-   -   Lanes 3 and 4, mAb179: a monoclonal antibody prepared against a        peptide corresponding to the C-terminus of Sub-1 (the large        subunit of the caspase-8, residues Cys360-Asp374).    -   Lanes 5 and 6, mAb 183.1 and lanes 7 and 8, mAb 183.2, two        monoclonal antibodies prepared against a peptide corresponding        to the N-terminus of Sub-1 (residues Ser217-Gly234).    -   Lanes 9 and 10, mAb 182 a monoclonal antibody prepared against a        peptide corresponding to the N-terminus of Sub-2 (the small        subunit of the caspase-8) (residues Lys385-Asp399).    -   Lane 11, NMS—normal mouse serum.

The figure shows Western blotting assessment of the amounts of caspase-8left in the cell lysates following immunoprecipitation by the indicatedantibodies and in total cell lysates (lanes 1 and 2).

FIG. 3 a shows the elution of the caspase-8 immunoprecipitated as inFIG. 2 by competing with the peptides against which the variousantibodies have been raised. Caspase-8 in the eluates from theimmunoprecipitates produced with the indicated antibodies is detected byWestern blot analysis (as in FIG. 2).

FIG. 3 b shows the elution of the caspase-8 immunoprecipitated as inFIG. 2 by competing with the peptides against which the variousantibodies have been raised. Caspase-8 in the eluates from theimmunoprecipitates produced with the indicated antibodies is shown bySilver staining.

FIG. 4 shows effective immunoprecipitation of minute amounts ofcaspase-8 found in lysates of BJAB cells using polyclonal serum preparedby immunization with a peptide corresponding to the C-terminus of Sub-1(the large subunit of the caspase-8, residues Cys360-Asp374). Depletionof caspase-8 from the BJAB cell lysates (prepared before, −, and after,+, Fas receptor stimulation) by immunoprecipitation with variousantibodies is shown from left to right. Caspase-8 left in the lysate isdetected by Western blot analysis after immunoprecipitation with thefollowing antibodies:

-   -   Lanes 3 and 4, NMS—normal mouse serum    -   Lanes 5 and 6, anti 179 polyclonal antibodies, a rabbit        polyclonal antibody prepared against the C-terminus of Sub-1        (the large subunit of the caspase-8, residues Cys360-Asp374).    -   Lanes 7 and 8 mAb 182.    -   TL-total cell lysate.

FIG. 5 shows immunoprecipitated and eluted caspase-8 from lysates ofnon-stimulated BJAB cells using various antibodies. Shown from left toright are the levels of caspase-8 detected by Western blot analysisafter elution of immunoprecipitates carried out with the followingantibodies:

-   -   Lane 1, anti 183 serum against the N-terminus of Sub-1 (residues        Ser217-Gly234).    -   Lane 2, mAb 183.2, a monoclonal antibody against the N-terminus        of Sub-1 (residues Ser217-Gly234).    -   Lane 3, mAb 179, a monoclonal antibody against the C-terminus of        Sub-1 (the large subunit of the caspase-8, residues        Cys360-Asp374).

The small (5.6 kDa) fragment of caspase-8, produced by thenovel-processing mode imposed by mAb 179 is marked with an arrow.

FIG. 6 shows caspase-8 and the caspase-8 bound polypeptide (p72/Cari)that had been immunoprecipitated by mAb 179 from lysates of BJAB cellsbefore or after one-hour stimulation with Fas-ligand and eluted bypeptide 179. Immunoprecipitated caspase-8 and bound polypeptides withmAb 179 were eluted (as in FIG. 3 b) resolved by SDS-PAGE and Silverstained. Lanes 1 and 2 show controls in which the cell lysates wereimmunoprecipitated with MIgG1, mouse immunoglobulin IgG1.

FIG. 7 shows a schematic representation of p72 (CARI) polypeptidemotifs. One coiled coil motif (C) and two tandem located ‘SURP motifs’(S) are located close to the N terminus of the polypeptide, and one‘G-patch’ motif is located at the C terminus of the polypeptide (Gmotif). The aspartic residue D600 present inside the G motif is alsoindicated. D600—a mutant in which residue D600 in the polypeptide wasreplaced with the glutamic acid residue.

FIG. 8 shows a schematic representation of the approach used for thefull-length preparation of p72 (Cari) cDNA. An EST clone IMAGE 2964545purchased from Incyte Genomics which lacks the sequence of the first 21nucleotides (which encode the first 7 amino acids) was used as thetemplate for a first polymerase chain reaction (PCR) together with apair of primers: the forward primer, P2 containing overlapping nucleicacids with the 5′ EST clone and additional 15 nucleotides out of the 21missing nucleotides and the reverse primer, P3 containing overlappingsequences with the 3′ EST. The resulting PCR product was used as atemplate for a second PCR together with a pair of primers: the forwardprimer, P1 containing the whole 21 missing nucleotides and 5 nucleicacids of the EST and the reverse primer, P3 containing overlappingsequences with the 3′ EST.

FIG. 9 shows co-immunoprecipitation of caspase-8 and p72 (Cari) byMab179 from the lysates of BJAB cells at time zero and after 20 minutesstimulation with Fas-ligand. The polypeptides eluted afterimmunoprecipitating with mAb 179 are resolved in SDS-PAGE gels anddetected by Silver staining. A band with an apparent molecular weight ofabout 72.5 kDa corresponding to p72 (Cari) is co-precipitated withpro-caspase-8 before Fas-ligand stimulation (lane 3). After 20 minutesof stimulation, the level of the 72.5 kDa band decreases and a new bandcorresponding to a polypeptide of lower apparent molecular weight ofabout 68 kDa is detected (lane 4).

Lane 1 and 2 show the negative controls comprising immunoprecipitationof cell lysates with MIgG1, mouse immunoglobulin IgG1.

FIG. 10 shows the cleavage of Cari by active caspase-8. A polypeptideencoded by p72 (Cari) cDNA was expressed in vitro in reticulocytelysates in the presence of ³⁵S methionine using the TnT T7 coupledreticulocyte lysate system, and tested after incubation of 1 hour at 37°C. in the presence or absence of recombinant active caspase-8. Inaddition the cleavage of Cari was studied with TnT products encoded by 2different p72 cDNA mutants: one encoding Cari in which the residue D600,suspected to be the target residue for caspase-8, was mutated to E (p72(D600E)) and another in which the gene is deleted and the resultingtruncated polypeptide lacks the residues down-stream (D600 p72 (1-600)).The resulting polypeptides were separated on SDS-PAGE and the resultswere visualized by phosphoimaging.

FIG. 11 shows caspase-8 and p72 (Cari) that were co-immunoprecipitatedby mAb 179 from the lysates of BJAB cells before (0′) or after 5, 10,20, 40 and 60 minutes of stimulation with Fas-ligand. The peptides wereeluted resolved in SDS-PAGE gels and detected by Silver staining. Apeptide of apparent molecular weight of 72.5 kDa is detected beforestimulation (0′). After 5 and 10 minutes of stimulation a newpolypeptide with a lower apparent molecular weight of about 68 kDaappears. After 40 minutes of stimulation the 72.5 kDa band completelydisappears and only the 68 kDa is detected. At 60 minutes none of theabove polypeptides mentioned were co-precipitated with caspase-8.

FIG. 12 a shows the effect of p72 (Cari) on apoptotic cell death inducedby the TNF receptor-signaling pathway. p72 (Cari) cDNA (p72) orantisense p72 (a/s) was inserted into the pcDNA 3.1 expression vectorand co-transfected with the p55 TNF receptor inserted in the pcDNA 3.1vector and with the green fluorescence protein (GFP) expressed from thepEGFPC1 vector, into HEK 293 cells constitutively expressing the Tantigen (as a negative control the vector without p72 cDNA insert wasused (pc)). After 24 hours, the transfected cells were examined under afluorescent microscope and cell death was scored by determining thenumber of cells displaying apoptotic morphology out of the totalpopulation of fluorescent cells.

FIG. 12 b shows induction of cell death by overexpression of p72 (Cari)in combination with Fas-ligand stimulation. The effect of Carioverexpression on Fas ligand mediated cell death was monitored in HEK293 cells constitutively expressing the T antigen. In the experiment thecells were co-transfected with a vector pcDNA3.1 (control group) or withpcDNA3.1 encoding Cari or its antisense (pc, p72 or p72 a/s,respectively) and a vector pSBC-2 encoding secreted alkaline phosphatase(SEAP). After 24 hours the transfected cells were induced withFas-ligand for 16 hours and the growth medium replaced with fresh growthmedium. Cell death was measured by determining the amount of SEAPsecreted into the growth medium in a period of the next 24 hours.

FIG. 13 shows the alignment between the sequence of the polypeptideobtained in the THC report (THC510568 SEQ ID NO:1) containing theconsensus of all the ESTs and the polypeptide predicted by the generatedfull-length cDNA (SEQ ID NO:3).

FIG. 14 shows kinetics of cell-death regulation by the Carinon-cleavable mutant D600E p72. Survival of BJAB cells was monitoredafter Fas ligand application in control cells (B1), cells constitutivelyexpressing transfected p72 (B2) and cells constitutively expressingtransfected D600E p72(B3).

FIG. 15 shows the minimal amino acid sequence (SEQ ID NO:5) of thepolypeptide in Cari responsible for binding caspase-8. Identification ofthe minimal polypeptide in Cari that is responsible for binding topro-caspase-8 was obtained by a detailed deletion study andco-precipitation with pro-caspase-8.

FIG. 16 shows inhibition of apoptosis by expression of a Cari antisensemolecule, pSuper-Cari. Apoptosis of cells was induced by overexpressionof caspase-8 (Mach a1) or a chimera of extracellular part of p55 TNF R1fussed to transmembrane and intracellular part of Fas receptor (Cl*) byseveral independent transfections carried out with vectors encodingthese polypeptides and inhibition by Cari antisense was assessed bycotransfection with pSuper-Cari (bars filled horizontally) or withpSuper-vector (Control, bars filled vertically).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an intracellular caspase bindingpolypeptide p72 (or Cari) or an isoform, a mutein, an allelic variant ora fragment thereof.

On one hand, Cari binds to pro-caspase-8 and enhances the conversion ofpro-caspase-8 into active caspase-8 and, on the other hand, activecaspase-8 cleaves Cari, and thus the activity of Cari is down-regulatedby the active caspase-8.

Cari may bind in addition to pro-caspase-8, to a different caspase, or amutein or fragment thereof and affect the activity of such caspase aswell.

In addition, the invention relates to a fragment of Cari or to a muteinhaving a dominant-negative effect on the activity of the endogenous Caripolypeptide and to a Cari polypeptide or a mutein or fragment capable ofincreasing the cytotoxic effect of a caspase, preferably, caspase-8.

For example, a non-cleavable Cari mutant (Cari D600E) polypeptide,wherein the aspartic acid 600 was replaced with a glutamic acid, wasgenerated. This polypeptide induces higher cytotoxicity than the wildtype version. Also, small peptides derived from Cari (24 and 16 aminoacid residues SEQ ID NO:4 and SEQ ID NO:5, respectively) containing thedomain in Cari that was shown to be responsible for binding caspase-8,were generated. Such peptides may inhibit binding of Cari topro-caspase-8 and therefore inhibit the cytotoxic effect of caspase-8.

For the identification of caspase bound polypeptides (e.g., Cari), cellscan be lysed before or after ligand stimulation (e.g., Fas ligand) andsubjected to immunoprecipitation by a suitable caspase specificantibody.

A suitable monoclonal antibody for the immunoprecipitation was generatedagainst a peptide from the C-terminal domain of caspase-8 Sub-1. Thisantibody was capable to immunoprecipitate pro-caspase-8 together withthe caspase-8-bound protein (e.g., Cari) and both the caspase-8 andcaspase-bound protein could be efficiently eluted from the immunecomplex and recovered in the supernatant by competing with a peptidederived from the caspase that was originally used to generate theantibodies.

Thus caspase bound polypeptides may be co-immunoprecipitated accordingto the invention, with such caspase specific suitable antibodies fromsamples selected from cell lysates of resting or stimulated cells, fromexpression cDNA libraries and from genomic or combinatorial peptidelibraries.

Stimulation of the cells can be effected by lymphokines, for example,Fas-ligand, TNF by environmental factors such as starvation, heat shocketc.

Antibodies may be developed against caspase bound proteins (e.g., Cari)found according to the invention. The antibodies specific to Cari,including the fragments thereof, may be used also to quantitatively orqualitatively detect Cari in a sample or to detect presence of cells,which express Cari. This can be accomplished by immunofluorescencetechniques employing a fluorescent labeled antibody (see below) coupledwith light microscopic, flow cytometric, or fluorometric detection.

The generation of polyclonal antibodies against polypeptides isdescribed Chapter 2 of Ausubel (1978-1995, 1999 and 2003). Thegeneration of antibodies against peptides may necessitate some changesin protocol, because of the generally lower antigenicity of peptideswhen compared to polypeptides. The generation of polyclonal antibodiesagainst peptides is described in Ausubel (1978-1995, 1999 and 2003),Chapter 9.

The antibodies prepared against Cari can be used for altering theactivity of the protein inside the cells, e.g., by selectively targetingCari on cells comprising transducing the cells with an intracellularlyexpressed antibody, or intrabody, against the Cari. The preparation ofintrabodies is disclosed in WO 99/14353.

Monoclonal antibodies may be prepared from B cells taken from the spleenor lymph nodes of immunized animals, in particular rats or mice, byfusion with immortalized B cells under conditions, which favors thegrowth of hybrid cells. For fusion of murine B cells, the cell line Ag-8is preferred.

The technique of generating monoclonal antibodies is described in manyarticles and textbooks, such as Ausubel (1978-1995, 1999 and 2003),Chapter 2. Chapter 9 therein describes the immunization, with peptides,or animals. Spleen or lymph node cells of these animals may be used inthe same way as spleen or lymph node cells of polypeptide-immunizedanimals, for the generation of monoclonal antibodies as described inchapter 2 therein.

The techniques used in generating monoclonal antibodies are furtherdescribed in Kohler and Milstein (1975), and in U.S. Pat. No. 4,376,110.

The preparation of antibodies from a gene bank of human antibodies thehyper variable regions thereof are replaced by almost random sequencesis described in U.S. Pat. No. 5,840,479. Such antibodies are preferredif it is difficult to immunize an animal with a given peptide orpolypeptide. Some structures are poorly immunogenic and may remain sodespite of the addition of adjuvants and of linking to otherpolypeptides in fusion constructs. The antibodies described in U.S. Pat.No. 5,840,479 are further preferred if it is desired to use antibodieswith a structure similar to human antibodies, for instance, whenantibodies are desired that have a low immunogenicity in humans.

Once a suitable antibody has been identified, it may be desired tochange the properties thereof. For instance, a chimeric antibody mayachieve higher yields in production. Chimeric antibodies wherein theconstant regions are replaced with constant regions of human antibodiesare further desired when it is desired that the antibody be of lowimmunogenicity in humans. The generation of chimeric antibodies isdescribed in a number of publications, such as Cabilly et al (1984),Morrison et al (1984), Boulianne et al (1984), EP 125023, EP 171496, EP173494, EP 184187, WO 86/01533, WO 87/02671, and Harlow and Lane (1988).

“Fully humanized antibodies” are molecules containing both the variableand constant region of the human immunoglobulin. Fully humanizedantibodies can be potentially used for therapeutic use, where repeatedtreatments are required for chronic and relapsing diseases such asautoimmune diseases. One method for the preparation of fully humanantibodies consist of “humanization” of the mouse humoral immune system,i.e., production of mouse strains able to produce human Ig (Xenomice),by the introduction of human immunoglobulin (Ig) loci into mice in whichthe endogenous Ig genes have been inactivated. The Ig loci areexceedingly complex in terms of both their physical structure and thegene rearrangement and expression processes required to ultimatelyproduce a broad immune response. Antibody diversity is primarilygenerated by combinatorial rearrangement between different V, D, and Jgenes present in the Ig loci. These loci also contain the interspersedregulatory elements, which control antibody expression, allelicexclusion, class switching and affinity maturation. Introduction ofunrearranged human Ig transgenes into mice has demonstrated that themouse recombination machinery is compatible with human genes.Furthermore, hybridomas secreting antigen specific hu-mAbs of variousisotypes can be obtained by Xenomice immunization with antigen.

Fully humanized antibodies and methods for their production are known inthe art (Mendez et al, 1997; Bruggemann et al, 1991; Tomizuka et al,2000; Patent WO 98/24893.

Another type of antibody is an anti-idiotypic antibody. Ananti-idiotypic (anti-Id) antibody is an antibody, which recognizesunique determinants generally associated with the antigen-binding siteof an antibody. An Id antibody can be prepared by immunizing an animalof the same species and genetic type (e.g., mouse strain) as the sourceof the mAb to which an anti-Id is being prepared. The immunized animalwill recognize and respond to the idiotypic determinants of theimmunizing antibody by producing an antibody to these idiotypicdeterminants (the anti-Id antibody). See, for example, U.S. Pat. No.4,699,880, which is herein entirely incorporated by reference.

The anti-Id antibody may also be used as an “immunogen” to induce animmune response in yet another animal, producing a so-calledanti-anti-Id antibody. The anti-anti-Id may be epitopically identical tothe original mAb, which induced the anti-Id. Thus, by using antibodiesto the idiotypic determinants of a mAb, it is possible to identify otherclones expressing antibodies of identical specificity.

The term “antibody” is also meant to include both intact molecules aswell as fragments thereof, such as, for example, Fab and F (ab′) 2,which are capable of binding antigen. Fab and F (ab′) 2 fragments lackthe Fc fragment of intact antibody, clear more rapidly from thecirculation, and may have less non-specific tissue binding than anintact antibody (Wahl et al, 1983).

It will be appreciated that Fab and F (ab′) 2 and other fragments of theantibodies useful in the present invention may be used for the detectionand quantitation of Cari according to the methods disclosed herein forintact antibody molecules. Such fragments are typically produced byproteolytic cleavage, using enzymes such as papain (to produce Fabfragments) or pepsin (to produce F (ab′) 2 fragments).

An antibody is said to be “capable of binding” a molecule if it iscapable of specifically reacting with the molecule to thereby bind themolecule to the antibody. The term “epitope” is meant to refer to thatportion of any molecule capable of being bound by an antibody, which canalso be recognized by that antibody. Epitopes or “antigenicdeterminants” usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and have specificthree-dimensional structural characteristics as well as specific chargecharacteristics.

The antibodies (or fragments thereof) useful in the present inventionmay be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of Cari. In situdetection may be accomplished by removing a histological specimen from apatient, and providing the labeled antibody of the present invention tosuch a specimen. The antibody (or fragment) is preferably provided byapplying or by overlaying the labeled antibody (or fragment) to abiological sample. Through the use of such a procedure, it is possibleto determine not only the presence of Cari polypeptide, but also itsdistribution on the examined tissue. Using the present invention, thoseof ordinary skill will readily perceive that any of wide variety ofhistological methods (such as staining procedures) can be modified inorder to achieve such in situ detection.

Such assays for the Cari polypeptide of the present invention typicallycomprises incubating a biological sample, such as a biological fluid, atissue extract, freshly harvested cells such as lymphocytes orleukocytes, or cells which have been incubated in tissue culture, in thepresence of a detectably labeled antibody capable of identifying theCari polypeptide, and detecting the antibody by any of a number oftechniques well known in the art.

“Biological fluid” or biological sample denotes any fluid derived fromor containing cells, cell components or cell products. Biological fluidsinclude, but are not limited to, cell culture supernatants, celllysates, cleared cell lysates, cell extracts, tissue extracts, blood,plasma, serum, milk and fractions thereof.

The biological sample may be treated with a solid phase support orcarrier such as nitrocellulose, or other solid support or carrier, whichis capable of immobilizing cells, cell particles or solublepolypeptides. The support or carrier may then be washed with suitablebuffers followed by treatment with a detectably labeled antibody inaccordance with the present invention, as noted above. The solid phasesupport or carrier may then be washed with the buffer a second time toremove unbound antibody. The amount of bound label on said solid supportor carrier may then be detected by conventional means.

By “solid phase support”, “solid phase carrier”, “solid support”, “solidcarrier”, “support” or “carrier” is intended any support or carriercapable of binding antigen or antibodies. Well-known supports orcarriers include glass, polystyrene, polypropylene, polyethylene,dextran, nylon amylases, natural and modified celluloses,polyacrylamides, gabbros and magnetite. The nature of the carrier can beeither soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen or antibody. Thus, the support or carrierconfiguration may be spherical, as in a bead, cylindrical, as in theinside surface of a test tube, or the external surface of a rod.Alternatively, the surface may be flat such as a sheet, test strip, etc.Preferred supports or carriers include polystyrene beads. Those skilledin the art will know may other suitable carriers for binding antibody orantigen, or will be able to ascertain the same by use of routineexperimentation.

The binding activity of a given lot of antibody, of the invention asnoted above, may be determined according to well-known methods. Thoseskilled in the art will be able to determine operative and optimal assayconditions for each determination by employing routine experimentation.

Other such steps as washing, stirring, shaking, filtering and the likemay be added to the assays as is customary or necessary for theparticular situation.

One of the ways in which an antibody in accordance with the presentinvention can be detectably labeled is by linking the same to an enzymeand used in an enzyme immunoassay (EIA). This enzyme, in turn, whenlater exposed to an appropriate substrate, will react with the substratein such a manner as to produce a chemical moiety, which can be detected,for example, by spectrophotometric, fluorometric or by visual means.Enzymes which can be used to detectably label the antibody include, butare not limited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholine-esterase. The detection can be accomplished bycalorimetric methods, which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

Detection may be accomplished using any of a variety of otherimmunoassays. For example, by radioactive labeling the antibodies orantibody fragments, it is possible to detect R-PTPase through the use ofa radioimmunoassay (RIA). A good description of RIA may be found inLaboratory Techniques and Biochemistry in Molecular Biology, by Work etal, North Holland Publishing Company, NY (1978) with particularreference to the chapter entitled “An Introduction to Radioimmune Assayand Related Techniques” by Chard T., incorporated by reference herein.The radioactive isotope can be detected by such means as the use of a gcounter or a scintillation counter or by autoradiography.

It is also possible to label an antibody in accordance with the presentinvention with a fluorescent compound. When the fluorescent-labeledantibody is exposed to light of the proper wavelength, its presence canbe then detected due to fluorescence. Among the most commonly usedfluorescent labeling compounds are fluorescein isothiocyanate,rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehydeand fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²E, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriamine pentaacetic acid (ETPA).

The antibody can also be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

An antibody molecule of the present invention may be adapted forutilization in an immunometric assay, also known as a “two-site” or“sandwich” assay. In a typical immunometric assay, a quantity ofunlabeled antibody (or fragment of antibody) is bound to a solid supportor carrier and a quantity of detectably labeled soluble antibody isadded to permit detection and/or quantitation of the ternary complexformed between solid-phase antibody, antigen, and labeled antibody.

Typical, and preferred, immunometric assays include “forward” assays inwhich the antibody bound to the solid phase is first contacted with thesample being tested to extract the antigen from the sample by formationof a binary solid phase antibody-antigen complex. After a suitableincubation period, the solid support or carrier is washed to remove theresidue of the fluid sample, including un-reacted antigen, if any, andthen contacted with the solution containing an unknown quantity oflabeled antibody (which functions as a “reporter molecule”). After asecond incubation period to permit the labeled antibody to complex withthe antigen bound to the solid support or carrier through the unlabeledantibody, the solid support or carrier is washed a second time to removethe un-reacted labeled antibody.

In another type of “sandwich” assay, which may also be useful with theantigens of the present invention, the so-called “simultaneous” and“reverse” assays are used. A simultaneous assay involves a singleincubation step as the antibody bound to the solid support or carrierand labeled antibody are both added to the sample being tested at thesame time. After the incubation is completed, the solid support orcarrier is washed to remove the residue of fluid sample and un-complexedlabeled antibody. The presence of labeled antibody associated with thesolid support or carrier is then determined as it would be in aconventional “forward” sandwich assay.

In the “reverse” assay, stepwise addition first of a solution of labeledantibody to the fluid sample followed by the addition of unlabeledantibody bound to a solid support or carrier after a suitable incubationperiod is utilized. After a second incubation, the solid phase is washedin conventional fashion to free it of the residue of the sample beingtested and the solution of un-reacted labeled antibody. Thedetermination of labeled antibody associated with a solid support orcarrier is then determined as in the “simultaneous” and “forward”assays.

The creation of immunoassays, such as RIA or ELISA, has been describedin many articles, textbooks, and other publications. Reference is madeto WO 97/03998, p. 48, line 4 to p. 52, line 27. Immunoassays of theinvention may be if two general types: Firstly, immunoassays usingimmobilized Cari polypeptide, or an equivalent peptide, may be used inthe quantification of Cari. Secondly, immunoassays using immobilizedantibodies directed against an epitope of a Cari polypeptide may be usedto quantify Cari polypeptides.

Such assays may find use in diagnostics, as the level of Cari and ofother polypeptides involved in apoptotic pathways may need to beevaluated in a number of disorders or syndromes where involvement ofsuch pathways is a possibility.

The terms protein and polypeptide are interchangeable in the presentspecification.

The polypeptide of the invention, a72 kDa polypeptide, was found tospecifically bind pro-Caspase-8. The polypeptide was then furtheranalyzed by partial sequencing and mass-spec analysis. The so-obtainedsequence was then entered into a database search program and overlappingsequences and ESTs were identified by computer search. The programs usedare well known to all of skill in the art and comprise, e.g., the GCG(genetics computer group) package. Preferably, a search utility such asBasic Local Alignment Search Tool (BLAST) available from the EMBL server(e.g., http://dove.embl-heidelberg.de/Blast2/) is used. The Blastncommand may be used for searching for nucleotide sequences that areoverlapping or similar with the clone identified.

Alternatively, or in addition to the above-noted methods of searchingdatabases, a library, such as a genomic library or a cDNA library, maybe screened in order to identify complete clones. Such screening methodsare described in the above-noted Sambrook et al (1989) and Ausubel et al(1978-1995, 1999 and 2003). Alternatively, or in addition, PCR-basedcloning techniques may be used, such as rapid amplification of cDNA ends(5′ and 3′ RACE, Graham et al, 1991, and references therein).

In the present embodiment the EST sequence found was searched in a TIGRHuman gene index and the THC report was obtained. Consensus of all theESTs, that fit these sequences, THC510568, was obtained (SEQ ID NO:1).The consensus sequence lacked the nucleotides that encode the firstmethionine and the subsequent 6 amino acids of Cari as judging from themouse EST that exhibits high similarity to the human EST (about 90%identity). The first methionine and the subsequent 6 amino acids of amouse counterpart protein, which were not missing in the mouse ESTs,were compared to the working draft sequence of the human genome in orderto complete the missing human sequence. A hit was obtained correspondingto the sequence of Homo sapiens chromosome 19, clone LLNLR-232E12. Thisclone confirmed the missing 7 amino acids of p72. The full-length cDNAof the p72 protein was obtained by PCR schematically represented in FIG.8. The whole DNA encoding p72 was recovered, sequenced (SEQ ID NO:2) andthe amino acid sequence deduced (SEQ ID NO:3).

P72 polypeptide was found to contain three conserved motifs (FIG. 7):the C motif a coiled motif, two tandem located ‘SURP’ (also called‘SWAP’ motifs, denoted as S FIG. 7) (Denhez and Lafyatis, 1994) close tothe N terminus of the polypeptide, and one C terminally located‘G-patch’ (FIG. 7 denoted as G) (Aravind and Koonin, 1999). Both theSURP and the G-patch motifs are believed to contribute to RNA-binding,suggesting that the target of p72 may be a RNA molecule. Thus p72 wasrenamed Cari (the name stands for Caspase-8 Associated polypeptide withRNA binding motifs). Thus, the terms Cari and p72 in the presentspecification are interchangeable.

The band corresponding to the full Cari polypeptide disappears afterstimulation of the cells and instead, a new polypeptide of lowermolecular weight appears. The possibility that Cari might be cleaved byactivated caspase-8 was inspected by an in vitro assay comprisingincubating recombinant produced caspase-8 and Cari labeled polypeptide.Cari may be produced by introducing the coding sequence thereof into anexpression vector containing a strong promoter and transfection into amammalian cell. Alternatively, Cari may be produced in vitro using an invitro translation system. The technique of in vitro translation is wellknown to the person of skill in the art, and reagents and detailedprotocols therefore are available, e.g., from Stratagene, La Jolla, USA.

In the present embodiment, Cari was labeled, using a radioisotope.Advantageously, when using isotopic labeling, the polypeptide to betested is expressed in vitro and the isotopically labeled amino acid,preferably, the isotope is S³⁵, together with unlabeled amino acid, isadded during the in vitro translation reaction. Further preferably, thelabeled amino acid is S³⁵-Methionine and the ration between libeled andunlabeled amino acid is 1:1 to about 1:1000.

The radioisotope labeled Cari polypeptide and the recombinant producedcaspase-8 active enzyme were then combined in a suitable buffer and fora time period sufficient to allow cleavage to occur. The preferredbuffer and other preferred parameters of the assay are described in thepublication Boldin et al (1996). The preferred time period is generallybetween 10 min and several hours, preferably between 30 min and onehour.

After allowing cleavage to occur, the reaction was resolved by SDSpolyacrylamide gel electrophoresis. The gel was dried and the isotopewas detected by photographic film or by phosphoimaging. The polypeptideto be tested can be tagged, and may be detected also by usingtag-specific antibodies in a Western blot.

The appearance of additional low molecular weight bands in reactions, inwhich caspase-8 is added, by comparison with control reactions withoutcaspase-8, indicates cleavage of Cari by caspase-8. The calculated sizeof the lower molecular weight band detected after the cleavage hints tothe approximate location of the cleavage site.

Mutation analysis studies were carried out to find the accurate residuetarget in Cari. Residue D-600 was found to be the target for cleavagesince a mutant Cari having a mutation in residue 600 from D to E is notcleaved by caspase-8 (p72/Cari D600E mutant).

The present invention relates also to the DNA sequence encoding Cari.Moreover, the present invention further concerns the DNA sequencesencoding a biologically active isoform, mutein, allelic variant,fragment or fusion protein of Cari. The preparation of such muteins andfragments and derivatives is by standard procedure (see for example,Sambrook et al, 1989) in which in the DNA sequences encoding Cari, oneor more codons may be deleted, added or substituted by another, to yieldmuteins having at least one amino acid residue change with respect tothe native polypeptide, except a mutein exhibiting Glycine at amino acidresidue 230 in place of Glutamic acid as in the polypeptide DF518-3 inWO 98/30582.

The DNA sequences of the invention encode Cari, isoform, allelicvariant, fragment, muteins, or derivative, DNA sequences capable ofhybridizing with a cDNA sequence derived from the coding region of anative Cari polypeptide, in which such hybridization is performed undermoderately stringent conditions, and which hybridizable DNA sequencesencode a biologically active Cari. These hybridizable DNA sequencestherefore comprise DNA sequences which have a relatively high similarityto the native Cari cDNA sequence and as such represent Cari-likesequences which may be, for example, naturally-derived sequencesencoding the various Cari isoforms, or naturally-occurring sequencesencoding polypeptides belonging to a group of Cari-like sequencesencoding a polypeptide having the activity of Cari. Further, thesesequences may also, for example, include non-naturally occurring,synthetically produced sequences, which are similar to the native CaricDNA sequence but incorporate a number of desired modifications. Suchsynthetic sequences therefore include all of the possible sequencesencoding muteins, fragments and derivatives of Cari, all of which havethe activity of Cari.

As used herein, stringency conditions are a function of the temperatureused in the hybridization experiment, the molarity of the monovalentcations and the percentage of formamide in the hybridization solution.To determine the degree of stringency involved with any given set ofconditions, one first uses the equation of Meinkoth et al (1984) fordetermining the stability of hybrids of 100% identity expressed asmelting temperature T_(m) of the DNA-DNA hybrid:T _(m)=81.5° C.+16.6(LogM)+0.41 (% GC)−0.61 (% form)−500/Lwhere M is the molarity of monovalent cations, % GC is the percentage ofG and C nucleotides in the DNA, % form is the percentage of formamide inthe hybridization solution, and L is the length of the hybrid in basepairs. For each 1° C. that the T_(m) is reduced from that calculated fora 100% identity hybrid, the amount of mismatch permitted is increased byabout 1%. Thus, if the T_(m) used for any given hybridization experimentat the specified salt and formamide concentrations is 10° C. below theT_(m) calculated for a 100% hybrid according to the equation ofMeinkoth, hybridization will occur even if there is up to about 10%mismatch.

“Moderately stringent conditions” are those which provide a T_(m) whichis not more than 20° C. below the T_(m) that would exist for a perfectduplex with the target sequence, either as calculated by the aboveformula or as actually measured. Without limitation, moderatelystringent (15-20° C. below the calculated or measured T_(m) of thehybrid) conditions use a wash solution of 2×SSC (standard salinecitrate) and 0.5% SDS (sodium dodecyl sulfate) at the appropriatetemperature below the calculated T_(m) of the hybrid. The ultimatestringency of the conditions is primarily due to the washing conditions,particularly if the hybridization conditions used are those, which allowless stable hybrids to form along with stable hybrids. The washconditions at higher stringency then remove the less stable hybrids. Acommon hybridization condition that can be used with moderatelystringent wash conditions described above is hybridization in a solutionof 6×SSC (or 6×SSPE) (standard saline-phosphate-EDTA), 5× Denhardt'sreagent, 0.5% SDS, 100 μg/ml denatures, fragmented salmon sperm DNA at atemperature approximately 200 to 25° C. below the T_(m). If mixed probesare used, it is preferable to use tetramethyl ammonium chloride (TMAC)instead of SSC (Ausubel, 1978-1995, 1999 and 2003).

To obtain the various above noted naturally occurring Cari-likesequences, standard procedures of screening and isolation ofnaturally-derived DNA or RNA samples from various tissues may beemployed using the natural Cari cDNA or portion thereof as probe (see,for example, standard procedures set forth in Sambrook et al, 1989).

The caspase binding polypeptide of the invention could be identified bythe above immunoprecipitation with mAb 179 specific to C-terminal domainof the Sub-1 of caspase-8. However in the above immunoprecipitationassay, antibody specific to the C-terminal domain of the Sub-1 from adifferent caspase than caspase-8 can be used in exchange. The inventionalso relates to a polypeptide or protein substantially corresponding toCari. The term “substantially corresponding” includes not only Caripolypeptide but also polypeptides or proteins that are muteins thereof.They may also comprise the corresponding “fusion proteins”, i.e.,polypeptides comprising Cari or a mutation thereof fused with anotherprotein and having a longer half-life in body fluids. CARI can thereforebe fused with another protein such as, for example, an immunoglobulin, ahigh molecular weight polymer, such as polyethylene glycol (PEG), or thelike.

Muteins that substantially correspond to Cari polypeptide are thosepolypeptides in which one or more amino acid of the caspase-8interacting protein's amino acid sequence has been replaced with anotheramino acid, deleted and/or inserted, except a mutein exhibiting Glycineat amino acid residue 230 in place of Glutamic acid and provided thatthe resulting polypeptide exhibits substantially the same or higherbiological activity as the Cari to which it corresponds.

In order to substantially correspond to Cari, the changes in thesequence of Cari, such as isoforms are generally relatively minor.Although the number of changes may be more than ten, preferably thereare no more than ten changes, more preferably no more than five, andmost preferably no more than three such changes. While any technique canbe used to find potentially biologically active polypeptides, whichsubstantially correspond to polypeptide Cari, one such technique is theuse of conventional mutagenesis techniques on the DNA encoding thepolypeptide, resulting in a few modifications. The polypeptidesexpressed by such clones can then be screened for their ability to bindto caspase-8 and/or to modulate caspase-8 activity inmodulation/mediation of the intracellular pathways noted above.

“Conservative” changes are those changes, which would not be expected tochange the activity of the polypeptide, and are usually the first to bescreened as these would not be expected to substantially change thesize, charge or configuration of the polypeptide and thus would not beexpected to change the biological properties thereof.

Conservative substitutions of Cari polypeptide include a mutein whereinat least one amino acid residue in the polypeptide has beenconservatively replaced by a different amino acid. Such substitutionspreferably are made in accordance with the following list as presentedin Table IA, which substitutions may be determined by routineexperimentation to provide modified structural and functional propertiesof a synthesized polypeptide molecule while maintaining the biologicalactivity characteristic of Cari polypeptide.

TABLE IA Original Exemplary Residue Substitution Ala Gly; Ser Arg LysAsn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Ala; Pro His Asn; GlnIle Leu; Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Tyr; Ile Phe Met;Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

Alternatively, another group of substitutions of Cari are those in whichat least one amino acid residue in the polypeptide has been removed anda different residue inserted in its place according to the followingTable IB. The types of substitutions, which may be made in thepolypeptide, may be based on analysis of the frequencies of amino acidchanges between a homologous polypeptide of different species, such asthose presented in Table 1-2 of Schulz et al (1979), and FIGS. 3-9 ofCreighton TE (1983). Based on such an analysis, alternative conservativesubstitutions are defined herein as exchanges within one of thefollowing five groups:

TABLE IB 1. Small aliphatic, nonpolar or slightly polar residues: Ala,Ser, Thr (Pro, Gly); 2. Polar negatively charged residues and theiramides: Asp, Asn, Glu, Gln; 3. Polar, positively charged residues: His,Arg, Lys; 4. Large aliphatic nonpolar residues: Met, Leu, Ile, Val(Cys); and 5. Large aromatic residues: Phe, Tyr, Trp.

The three amino acid residues in parentheses above have special roles inprotein architecture. Gly is the only residue lacking any side chain andthus imparts flexibility to the chain. This, however, tends to promotethe formation of secondary structure other than a-helical. Pro, becauseof its unusual geometry, tightly constrains the chain and generallytends to promote beta-turn-like structures, although in some cases Cyscan be capable of participating in disulfide bond formation, which isimportant in protein folding. Note that Schulz et al (1979) would mergeGroups 1 and 2, above. Note also that Tyr, because of its hydrogenbonding potential, has significant kinship with Ser, and Thr, etc.

Conservative amino acid substitutions according to the presentinvention, e.g., as presented above, are known in the art and would beexpected to maintain biological and structural properties of thepolypeptide after amino acid substitution. Most deletions andsubstitutions according to the present invention are those, which do notproduce radical changes in the characteristics of the protein orpolypeptide molecule. “Characteristics” is defined in a non-inclusivemanner to define both changes in secondary structure, e.g., a-helix orbeta-sheet, as well as changes in biological activity, e.g., binding tocaspase-8 and/or mediation of the effect of caspase-8 on cell death.

Examples of production of amino acid substitutions in proteins which canbe used for obtaining muteins of caspase-8 interacting polypeptide Carifor use in the present invention include any known method steps, such aspresented in U.S. Pat. Nos. RE 33,653, 4,959,314, 4,588,585 and4,737,462 to Mark et al; U.S. Pat. No. 5,116,943 to Koths et al, U.S.Pat. No. 4,965,195 to Namen et al; U.S. Pat. No. 4,879,111 to Chong etal; and U.S. Pat. No. 5,017,691 to Lee et al; and lysine substitutedproteins presented in U.S. Pat. No. 4,904,584 to Shaw et al

Besides conservative substitutions discussed above which would notsignificantly change the activity of polypeptide Cari, eitherconservative substitutions or less conservative and more random changes,which lead to an increase in biological activity of the muteins of Caripolypeptide, are intended to be within the scope of the invention.

When the exact effect of the substitution or deletion is to beconfirmed, one skilled in the art will appreciate that the effect of thesubstitution(s), deletion(s), etc., will be evaluated by routine bindingand cell death assays. Screening using such a standard test does notinvolve undue experimentation.

Acceptable Cari muteins are those, which retain at least the capabilityof interacting with pro-caspase-8, and thereby, regulate the activity ofcaspase-8 in the intracellular pathways. In one embodiment Cari wasfound to increase cell death mediated by Fas, probably by increasing therate of pro-caspase-8 conversion into active caspase-8. Once caspase-8is formed it cleaves Cari causing probably its inactivation. Anon-cleavable mutein of Cari (Cari D600E mutant) was generated and foundto be more potent than the wild type polypeptide in cell deathinduction. Non-cleavable mutants and preferably the p72 D600E mutant canbe used in certain situations where it may be desired to increasecaspase-8 activity. Mutein polypeptides can be produced which have aso-called dominant-negative effect, namely, a polypeptide which isdefective either in binding to caspase-8, or in subsequent signaling orother activity following such binding. Muteins can be used, for example,to inhibit the cytotoxic effect of caspase-8, or to increase it,depending on whether it is desired to increase cell death or cellsurvival and depending on which of these activities is the major onemodulated by the interaction of Cari and caspase-8 (see above), and thisby such muteins competing with the natural Cari polypeptide for bindingto or interacting with caspase-8.

At the genetic level, muteins are generally prepared by site-directedmutagenesis of nucleotides in the DNA encoding the Cari polypeptide,thereby producing DNA encoding the mutein, and thereafter synthesizingthe DNA and expressing the polypeptide in recombinant cell culture. Themuteins typically exhibit the same or increased qualitative biologicalactivity as the naturally occurring polypeptide, Ausubel et al(1978-1995, 1999 and 2003); Sambrook et al (1989).

Preparation of a Cari in accordance herewith, or an alternativenucleotide sequence encoding the same polypeptide but differing from thenatural sequence due to changes permitted by the known degeneracy of thegenetic code, can be achieved by site-specific mutagenesis of DNA thatencodes an earlier prepared muteins or a native version of a Caripolypeptide. Site-specific mutagenesis allows the production of muteinsthrough the use of specific oligonucleotide sequences that encode theDNA sequence of the desired mutation, as well as a sufficient number ofadjacent nucleotides, to provide a primer sequence of sufficient sizeand sequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 20 to 25nucleotides in length is preferred, with about 5 to 10 complementingnucleotides on each side of the sequence being altered. In general, thetechnique of site-specific mutagenesis is well known in the art, asexemplified by publications such as Adelman et al (1983), the disclosureof which is incorporated herein by reference.

As will be appreciated, the site-specific mutagenesis techniquetypically employs a phage vector that exists in both a single-strandedand double-stranded form. Typical vectors useful in site-directedmutagenesis include vectors such as the M13 phage, for example, asdisclosed by Messing et al (1981), the disclosure of which isincorporated herein by reference. These phages are readily availablecommercially and their use is generally well known to those skilled inthe art. Alternatively, plasmid vectors that contain a single-strandedphage origin of replication (Vieira et al, 1987) may be employed toobtain single-stranded DNA.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector that includeswithin its sequence a DNA sequence that encodes the relevantpolypeptide. An oligonucleotide primer bearing the desired mutatedsequence is prepared synthetically by automated DNA/oligonucleotidesynthesis. This primer is then annealed with the single-strandedprotein-sequence-containing vector, and subjected to DNA-polymerizingenzymes such as E. coli polymerase I Klenow fragment, to complete thesynthesis of the mutation-bearing strand. Thus, a mutated sequence andthe second strand bear the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli JM101 cells,and clones are selected that include recombinant vectors bearing themutated sequence arrangement.

After such a clone is selected, the mutated Cari sequence may be removedand placed in an appropriate vector, generally a transfer or expressionvector of the type that may be employed for transfection of anappropriate host.

Accordingly, gene or nucleic acid encoding for Cari can also bedetected, obtained and/or modified, in vitro, in situ and/or in vivo, bythe use of known DNA or RNA amplification techniques, such as polymerasechain reaction (PCR) and chemical oligonucleotide synthesis. PCR allowsfor the amplification (increase in number) of specific DNA sequences byrepeated DNA polymerase reactions. This reaction can be used as areplacement for cloning; all that is required is knowledge of thenucleic acid sequence. In order to carry out PCR, primers are designedwhich are complementary to the sequence of interest. The primers arethen generated by automated DNA synthesis. Because primers can bedesigned to hybridize to any part of the gene, conditions can be createdsuch that mismatches in complementary base pairing can be tolerated.Amplification of these mismatched regions can lead to the synthesis of amutagenized product resulting in the generation of a peptide with newproperties (i.e., site directed mutagenesis). See also, e.g., Ausubel(1978-1995, 1999 and 2003), Chapter 16.

Furthermore, PCR primers can be designed to incorporate new restrictionsites or other features such as termination codons at the ends of thegene segment to be amplified. This placement of restriction sites at the5′ and 3′ ends of the amplified gene sequence allows for gene segmentsencoding Cari polypeptide or a fragment thereof to be custom designedfor ligation other sequences and/or cloning sites in vectors.

PCR and other methods of amplification of RNA and/or DNA are well knownin the art and can be used according to the present invention withoutundue experimentation, based on the teaching and guidance presentedherein. Known methods of DNA or RNA amplification include, but are notlimited to polymerase chain reaction and related amplification processes(see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188,to Mullis et al; U.S. Pat. Nos. 4,795,699 and 4,921,794 to Tabor et al;U.S. Pat. No. 5,142,033 to Innis; U.S. Pat. No. 5,122,464 to Wilson etal; U.S. Pat. No. 5,091,310 to Innis; U.S. Pat. No. 5,066,584 toGyllensten et al; U.S. Pat. No. 4,889,818 to Gelfand et al; U.S. Pat.No. 4,994,370 to Silver et al; U.S. Pat. No. 4,766,067 to Biswas; U.S.Pat. No. 4,656,134 to Ringold; and Inniset al, 1990) and RNA mediatedamplification which uses anti-sense RNA to the target sequence as atemplate for double stranded DNA synthesis (U.S. Pat. No. 5,130,238 toMalek et al, with the tradename NASBA); and immuno-PCR which combinesthe use of DNA amplification with antibody labeling (Ruzicka et al(1993); Sano et al (1992); Sano et al (1991), the entire contents ofwhich patents and reference are entirely incorporated herein byreference.

In an analogous fashion, biologically active fragments of Cari (e.g.,those of any of the Cari polypeptides or its isoforms) may be preparedas noted above with respect to the muteins of Cari. Suitable fragmentsof Cari are those which retain the pro-caspase-8 binding proteincapability and which can mediate the biological activity of Cari orother proteins associated with caspase-8 directly or indirectly.Alternatively, suitable fragments of Cari are those which retain thepro-caspase-8 binding protein capability and which can inhibit thebiological activity of Cari or other proteins associated with caspase-8directly or indirectly. Accordingly, Cari fragments can be preparedwhich have a dominant-negative or a dominant-positive effect as notedabove with respect to the muteins. It should be noted that thesefragments represent a special class of the muteins of the invention,namely, they are defined portions of Cari derived from the full Carisequence (e.g., from that of any one of the Cari protein or itsisoforms), each such portion or fragment having any of the above-noteddesired activities. Such fragment may be, e.g., a peptide.

Similarly, derivatives may be prepared by standard modifications of theside groups of one or more amino acid residues of the Cari polypeptide,its muteins or fragments, or by conjugation of the Cari polypeptide, itsmuteins or fragments, to another molecule, e.g., an antibody, enzyme,receptor, etc., as are well known in the art. Accordingly, “derivatives”as used herein covers derivatives which may be prepared from thefunctional groups which occur as side chains on the residues or the N-or C-terminal groups, by means known in the art, and are included in theinvention. Derivatives may have chemical moieties such as carbohydrateor phosphate residues, provided such a derivative has the same or higherbiological activity as Cari polypeptide.

For example, derivatives may include aliphatic esters of the carboxylgroups, amides of the carboxyl groups by reaction with ammonia or withprimary or secondary amines, N-acyl derivatives or free amino groups ofthe amino acid residues formed with acyl moieties (e.g., alkanoyl orcarbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl group(for example, that of seryl or threonyl residues) formed with acylmoieties.

The term “derivatives” is intended to include only those derivativesthat do not change one amino acid to another of the twenty commonlyoccurring natural amino acids.

As described above, the cleavage assays may be used to determine whetherCari polypeptide and muteins are cleaved by caspase-8.

In an embodiment of the present invention, the cleavage site (D600) wasfurther determined by preparing deletion mutants or point mutations ofCari and testing each deletion and point mutant for its susceptibilityto cleavage by caspase-8 as described above. Deletion mutants may beconstructed by PCR cloning of desired fragments of the polypeptide to betested, using the DNA sequence of the clone coding for said polypeptideto be tested as a template. The PCR amplified fragments may then beinserted into expression vectors, whereby an ATG start codon andpreferably, a Kozak sequence (Kozak, 1984) must be provided. Furtherdetails on expressing polypeptides may be found in the above-notedinformation of Qiagen, relating to his-tagged proteins, but also toprotein expression in general. Another reference for protein expressionof the further above-mentioned Ausubel (1978-1995, 1999 and 2003), andspecifically Chapter 16 therein.

The cleavage site of a polypeptide to be tested may thus be defined bypreparing various deletion mutants therefrom and determining thesmallest such deletion mutant that is cleaved by caspase-8.

Another way of identifying the cleavage site uses peptides, which aregenerated according to the predicted polypeptide sequence of the cloneto be tested. Peptides may be synthesized chemically, e.g., as detailedin Bodanszky and Bodanszky (1984), and Bodanszky (1993). Custom peptidesynthesis is further available from several commercial companies, e.g.,SynPep Corp., Dublin, Calif. USA, and California Peptide Research, Inc.,Napa, Calif., USA. Peptides may also be produced, either as fusion withother proteins or unfused, by expressing recombinant DNA codingtherefore, as detailed in Chapter 16 of Ausubel (1978-1995, 1999 and2003).

In order to use peptides for mapping the cleavage site of a polypeptideto be tested, the predicted amino acid sequence of said polypeptide isdivided into areas and a peptide corresponding to each area issynthesized. In addition, peptides comprising about half of the aminoacids of one area and contiguously comprising further about half of theamino acids of a directly neighboring area are synthesized, so as to beoverlapping the border between the two areas. The areas comprise between5 and 100 amino acids, preferably between 9 and 40 amino acids, and mostpreferably between 20 and 30 amino acids. After the cleavage reaction,they may therefore be analyzed directly by SDS polyacrylamide gelelectrophoresis and UV detection or visualization by staining, e.g.,using Coomassie blue. Alternatively, peptides may be labeled for easierdetection, e.g., by isotopic end labeling (see, e.g., Shevchenko et al,1997).

After a peptide screen as described above has been completed, thepeptide which is now known to comprise the cleavage site for caspase-8can be further studied be repeating the same technique, but choosingsmaller areas selected from the sequence of the peptide that has beenidentified.

The actual cleavage site of the peptides should conform to the caspasecleavage sequence XXXD (see Boldin et al, 1996 and Nicholson et al,1997). The contribution of each amino acid in the peptide may beevaluated by preparing peptides that are mutated in one amino acid andtesting these mutated peptides for susceptibility to cleavage withcaspase-8. The amino acid to be mutated is preferably replaced by anamino acid selected from the group of charged non-polar amino acids (seeLehninger,), most preferably selected from glycine or alanine.

By mutating critical amino acids, it is possible to generate peptidesthat bind pro-caspase-8, but are not susceptible to cleavage thereby.Binding may be tested by size separation of peptide-caspase-8 complexesunder non-denaturing conditions using acrylamide gel electrophoresis orby co-precipitation with caspase-8 specific antibodies.

The polypeptide to be tested, or a peptide fragment thereof, may befurther characterized by introducing said polypeptide or peptide into amammalian cell and measuring the effect of apoptosis-including reagentsin said cell.

Expression of a Cari polypeptide or peptide in a mammalian cell may bedone by inserting the DNA coding for Cari into a vector comprising apromoter, optionally an intron sequence and splicing donor/acceptorsignals, and further optionally comprising a termination sequence. Thesetechniques are in general described in Ausubel (1978-1995, 1999 and2003), Chapter 16.

The above promoter, intron, and termination sequences are operable inmammalian cells. The promoter is preferably a strong promoter such asthe above-noted RSV, CMV, or MPSV promoter. The promoter may also be theSV40 early promoter (Everett, et al, 1983, and references therein), or acellular promoter, such as the beta-actin promoter or the ELF-1 promoter(Tokushige et al, 1997). Also, a hybrid promoter may be used, such asthe hybrid between the lac operator and the human ELF-1 alpha promoteras described by Edamatsu et al, 1997, the CMV-beta actin hybrid promoterdescribed by Akagi et al (1997), or the hybrid between tet operatorsequences and the CMV promoter (Furth et al, 1994, and referencestherein).

Intron sequences, which may be inserted as complete sequences, i.e.,including the splice donor and acceptor sites, may be inserted into thecoding sequence of the polypeptide, which it is desired to express.Insertion if such intron sequences may enhance RNA stability and thusenhance production of the desired polypeptide. While, in principle,suitable intron sequences may be selected from any gene containingintrons, preferred intron sequences are the beta-actin intron, the SV 40intron, and the p55 TNF receptor intron.

The intron sequence may contain enhancer elements, which may enhancetranscription from the above-noted promoters.

Often, intron sequences also contain transcriptional or translationalcontrol sequences that confer tissue specific expression. Therefore,when it is desired to express a polypeptide of the invention in atissue-specific manner, such intron sequences may be advantageouslyemployed. An example of an intron containing tissue-specific enhancerelements is the erythroid-specific enhancer located in intron 8 of thehuman 5-aminolevulinate synthase 2 gene (Surinya et al, 1998), and adiscussion of the principle of enhancing protein production using intronsequences, together with example intron sequences, is provided in Huanget al, 1990.

Transcriptional termination sequences and polyadenylation signals may beadded at the 3′ end of the DNA coding for the polypeptide that it isdesired to express. Such sequences may be found in many or even mostgenes. Advantageously, the SV 40 polyadenylation signal can be used(Schek et al, 1992, and references therein).

Vectors for expression of Cari in a mammalian cell could be used, forexample, the pcDNA3.1 vector (Invitrogen), which contains the CMVpromoter for driving expression of the gene encoding the desiredpolypeptide and pMPSVEH vectors with the MPSV promoters.

Recombinant polypeptides can be produced either in bacterial oreukaryotic (e.g., CHO) cultured host cells transfected with vectorsencoding such polypeptides or in transgenic animals. When usingtransgenic animals, it is particularly advantageous to produceheterologous polypeptides in their milk. Dairy animals such as cattle,sheep and goats are thus preferred hosts. See, for example, patentspecifications WO 88/00239, WO 90/05188, WO 91/02318, and WO 92/11757;and U.S. Pat. Nos. 4,873,191; 4,873,316; and 5,304,489, which areincorporated herein by reference in their entirety.

Using recombinant expression of the polypeptide to be tested, thepolypeptide can now be evaluated for its effect on the apoptotic signal,which is mediated by a caspase, for example, caspase-8. For thatpurpose, apoptosis may be induced by either overexpression of anapoptosis-inducing protein, such as the p55 TNFR, the Mort-1 protein,caspase-8, or an equivalent thereof; or activation of an apoptoticsignal by triggering p55 TNFR, CD120a, CD95, TRAMP/DR3, or an equivalentreceptor. In one embodiment, apoptosis is induced by overexpression ofp55 TNFR.

Receptor activation may be achieved also by contacting the receptorswith specific ligands or by cross-linking receptors with antibodies,preferably polyclonal antibodies (see Engelmann et al, 1990). In oneembodiment overexpression of Cari is followed by stimulation withFas-ligand.

While in general, triggering of a receptor like CD95 by Fas Ligandrequires the addition of a protein synthesis inhibitor likecycloheximide in order to achieve a strong signal for apoptosis, theoverexpression of receptor intracellular domains or of proteins involvedin apoptosis signal transduction do not (see Boldin et al, 1996). Incontrast, when Fas Ligand stimulation was given to cells overexpressingCari, cycloheximide was not required to achieve a strong signal forapoptosis. The detection of apoptosis, incubation times and otherdetails and parameters for this assay have been described in the aboveBoldin et al.

Cell death in cells overexpressing Cari, versus control cells, may beevaluated by any number of methods, such as methods based upon DNAfragmentation or detection of apoptosis-specific antigens and epitopes.Reagents and protocols for detection of apoptosis in kit form areavailable from the above-noted Boehringer Mannheim and other companies.

Cell death may also be determined by evaluating the morphologicalappearance of the cells. Apoptotic cell death is characterized by a wavycell membrane and shrinking of the cells in the absence of cell lysis.

Advantageously, a reporter gene is expressed in the mammalian cell, inorder to provide a marker for successful transfection. As thetransfection procedure by itself results in some cell death, includingcell death of cells that have not been transfected, it is of advantageto only evaluate cells that have been transfected. A preferred reportergene for this purpose is the GFP, the green fluorescent protein, may beused for direct detection without the need for a color reaction, thisreporter gene necessitates the use of a fluorescent microscope. However,any other known reporter gene may be used, preferably a gene whoseproducts are easily detected using a simple color reaction, for example,lacZ gene, is easily detected by incubation of transfected cells withXgal or a similar reagent indicative of active beta-galactosidase theresults of which may be evaluated by using a microscope.

Thus, by only considering cells that have been transfected, i.e., thatexpress the reporter gene, and by counting the percentage of cellsdemonstrating apoptotic morphology, it is possible to evaluate theeffect of a particular transfected clone and the polypeptide expressedtherefrom on apoptosis.

Mammalian cells to be used for transfection and testing of apoptosis areselected from HeLa cells, human Caucasian chronic myelogenous withlymphoblast morphology (K562), human T cell lymphoma with lymphoblastmorphology (Hut78), human Negroid Burkitt's lymphoma with lymphoblastmorphology (Raji), Namalwa-human Burkitt's lymphoma with lymphoblastmorphology (Nalm), human Caucasian promyelocytic leukemia withpromyeloid morphology (HL-60), acute lymphoblastic leukemia withlymphoblast morphology (CEM) and human T cell with lymphoblastmorphology (H9) and preferably human embryonic kidney (HEK) 293 cellsoverexpressing the T antigen cells. The transfection is preferably doneby the calcium phosphate method as described in Ausubel (1978-1995, 1999and 2003). The morphology of the cells if evaluated one to 150 hoursafter transfection, preferably 4 to 35 hours and most preferably 24hours after transfection.

The use of a vector for inducing and/or enhancing the endogenousproduction of Cari or an inhibitor of Cari normally silent, are alsocontemplated according to the invention. The vector may compriseregulatory sequences functional in the cells desired to express Cari orthe inhibitor of Cari. Such regulatory sequences may be, for example,promoters or enhancers. The regulatory sequence may then be introducedinto the right locus of the genome by homologous recombination, thusoperably linking the regulatory sequence with the gene, the expressionof which is required to be induced or enhanced. The technology isusually referred to as “endogenous gene activation” (EGA), and it isdescribed, e.g., in WO 91/09955.

It will be understood by the person skilled in the art that it is alsopossible to shut down Cari expression using the same technique, i.e., byintroducing a negative regulation element, like, e.g., a silencingelement, into the gene locus of Cari, thus leading to down-regulation orprevention of Cari expression. The person skilled in the art willunderstand that such down-regulation or silencing of Cari expression hasthe same effect as the use of a Cari inhibitor in order to preventand/or treat disease.

The clones obtained in the screening of caspase binding polypeptides bythe method of the invention may be partial clones. The generation ofcomplete clones, if necessary, has been described further above. The DNAsequence of a complete clone and of the partial clone initially found inthe screening of the invention may find a variety of uses.

For example, in order to manipulate the expression of Cari, it may bedesirable to produce antisense RNA in a cell. For this purpose, thecomplete or partial cDNA, preferably 9 nucleotides, coding for Caripolypeptide is inserted into an expression vector comprising a promoter,as noted further above. The 3′ end of the cDNA is thereby insertedadjacent to the 3′ end of the promoter, with the 5′ end of the cDNAbeing separated from the 3′ end of the promoter by said cDNA. Uponexpression of the cDNA in a cell, an antisense RNA is therefore producedwhich is incapable of coding for the polypeptide. The presence ofantisense RNA in the cell reduces the expression of the cellular(genomic) copy of the Cari.

For the production of antisense RNA, the complete cDNA may be used.Alternatively, a fragment thereof may be used, which is preferablybetween about 9 and 2,000 nucleotides in length, more preferably between15 and 500 nucleotides, and most preferably between 20 and 150nucleotides.

A synthetic oligonucleotide may be used as antisense oligonucleotide.The oligonucleotide is preferably a DNA oligonucleotide. The length ofthe antisense oligonucleotide is preferably between 9 and 150, morepreferably between 12 and 60, and most preferably between 20 and 50nucleotides, for example, the sequence in SEQ ID NO:6 and SEQ ID NO:7.

The mechanism of action of antisense RNA and the current sate of the artof use of antisense tools is reviewed in Kumar et al (1998). The use ofantisense oligonucleotides in inhibition of BMP receptor synthesis hasbeen described by Yeh et al (1998). The use of antisenseoligonucleotides for inhibiting the synthesis of the voltage-dependentpotassium channel gene Kv1.4 has been described by Meiri et al (1998).The use of antisense oligonucleotides for inhibition of the synthesis ofBcl-x has been described by Kondo et al (1998).

The therapeutic use of antisense drugs is discussed by Stix (1998),Flanagan, (1998), Guinot and Temsamani, (1998), and references therein.

Modifications of oligonucleotides that enhance desired properties aregenerally used when designing antisense oligonucleotides. For instance,phosphorothioate bonds are used instead of the phosphoester bondsnaturally occurring in DNA, mainly because such phosphorothioateoligonucleotides are less prone to degradation by cellular enzymes. Penget al teach that undesired in vivo side effects of phosphorothioateoligonucleotides may be reduced when using a mixedphosphodiester-phosphorothioate backbone. Preferably,2′-methoxyribonucleotide modifications in 60% of the oligonucleotide areused. Such modified oligonucleotides are capable of eliciting anantisense effect comparable to the effect observed with phosphorothioateoligonucleotides. Peng et al (2001) teach further that oligonucleotidemuteins incapable of supporting ribonuclease H activity are inactive.

Therefore, the preferred antisense oligonucleotide of the invention hasa mixed phosphodiester-phosphorothioate backbone. Most preferably,2′-methoxyribonucleotide modifications in about 30% to 80%, mostpreferably about 60% of the oligonucleotide are used.

Further modification may be introduced to an antisense oligonucleotide.For instance, the oligonucleotide molecule may be linked to a groupcomprising optionally partially unsaturated aliphatic hydrocarbon chainand one or more polar or charged groups such as carboxylic acid groups,ester groups, and alcohol groups. Alternatively, oligonucleotides may belinked to peptide structures, which are preferably membranotropicpeptides. Such modified oligonucleotide penetrates membranes moreeasily, which is critical for their function and may thereforesignificantly enhance their activity. Membrane permeability isespecially desirable for antisense drugs that are desired to reach thebrain. Palmityl-linked oligonucleotides have been described by Gersteret al (1998). Geraniol-linked oligonucleotides have been described byShoji et al, (1998). Oligonucleotides linked to peptides, e.g.,membranotropic peptides, and their preparation have been described bySoukchareun et al, (1998). Modifications of antisense molecules or otherdrugs that target the molecule to certain cells and enhance uptake ofthe oligonucleotide by said cells are described by Wang (1998).

Given the known mRNA sequence of a gene, ribozymes may be designed,which are RNA molecule that specifically bind and cleave said mRNAsequence (see, e.g., Chen et al, 1992, Zhao and Pick 1993, Shore et al,1993, Joseph and Burke, 1993, Shimayama et al, 1993, Cantor et al,1993).

Accordingly, ribozyme-encoding RNA sequence may be designed that cleavethe mRNA of a Cari polypeptide of the invention. The point of cleavageis preferably located in the coding region or in the 5′ non-translatedregion, more preferably, in the 5′ part of the coding region close tothe AUG translation start codon.

A DNA encoding a ribozyme according to the invention may be introducedinto cells by way of DNA uptake, uptake of modified DNA (seemodifications for oligonucleotides and proteins that result in enhancedmembrane permeability, as described herein below), or viralvector-mediated gene transfer as detailed herein below.

The present invention provides therefore Cari, peptides derivedtherefrom, mutants, specific antibodies, DNA encoding the protein,ribozyme, antisense DNA molecules, and oligonucleotides. A therapeuticor research-associated use of these tools necessitates theirintroduction into cells of a living organism. For this purpose, it isdesired to improve membrane permeability of peptides, polypeptides andoligonucleotides. Derivatization with lipophilic structures may be usedin creating peptides and polypeptides with enhanced membranepermeability. For instance, the sequence of a known membranotropicpeptide as noted above may be added to the sequence of the peptide orpolypeptide. Further, the peptide or polypeptide may be derivatized bypartly lipophilic structures such as the above-noted hydrocarbon chains,which are substituted with at least one polar or charged group. Forexample, lauroyl derivatives of peptides have been described byMuranishi et al (1991). Further modifications of peptides andpolypeptides comprise the oxidation of methionine residues to therebycreate sulfoxide groups, as described by Zacharia et al (1991). Zachariaand co-workers also describe peptide or derivatives wherein therelatively hydrophobic peptide bond is replaced by its ketomethyleneisoester (COCH₂). These and other modifications known to the person ofskill in the art of polypeptide and peptide chemistry enhance membranepermeability.

Another way of enhancing membrane permeability is the use receptors,such as virus receptors, on cell surfaces in order to induce cellularuptake of the peptide or polypeptide. This mechanism is used frequentlyby viruses, which bind specifically to certain cell surface molecules.Upon binding, the cell takes the virus up into its interior. The cellsurface molecule is called a virus receptor. For instance, the integrinmolecules CAR and AdV have been described as virus receptors forAdenovirus, see Hemmi et al (1998), and references therein. The CD4,GPR1, GPR15, and STRL33 molecules have been identified asreceptors/co-receptors for HIV, see Edinger et al (1998) and referencestherein.

Thus, conjugating peptides, polypeptides or oligonucleotides tomolecules that are known to bind to cell surface receptors will enhancemembrane permeability of said peptides, polypeptides oroligonucleotides. Examples for suitable groups for forming conjugatesare sugars, vitamins, hormones, cytokines, transferrin,asialoglycoprotein, and the like molecules. Low et al U.S. Pat. No.5,108,921, describes the use of these molecules for the purpose ofenhancing membrane permeability of peptides, polypeptides andoligonucleotides, and the preparation of said conjugates.

Low and co-workers further teach that molecules such as folate or biotinmay be used to target the conjugate to a multitude of cells in anorganism, because of the abundant and unspecific expression of thereceptors for these molecules.

The above use of cell surface proteins for enhancing membranepermeability of a peptide, polypeptide or oligonucleotide of theinvention may also be used in targeting said peptide, polypeptide oroligonucleotide of the invention to certain cell types or tissues. For(instance, if it is desired to target cancer cells, it is preferable touse a cell surface protein that is expressed more abundantly on thesurface of those cells. Examples are the folate receptor, the mucinantigens MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, and MUC7, theglycoprotein antigens KSA, carcinoembryonic antigen, prostate-specificmembrane antigen (PSMA), HER-2/neu, and human chorionicgonadotropin-beta. The above-noted Wang et al (1998), teaches the use offolate to target cancer cells, and Zhang et al (1998), teaches therelative abundance of each of the other antigens noted above in varioustypes of cancer and in normal cells.

The polypeptide, peptide or oligonucleotide of the invention maytherefore, using the above-described conjugation techniques, be targetedto certain cell type as desired. For instance, if it is desired toenhance apoptosis in cells of the lymphocytic lineage, Cari peptide,fragment thereof, mutants and derivatives of the invention may betargeted at such cells, for instance, by using the MHC class IImolecules that are expressed on these cells. This may be achieved bycoupling an antibody, or the antigen-binding site thereof, directedagainst the constant region of said MHC class II molecule to thepolypeptide or peptide of the invention. Further, numerous cell surfacereceptors for various cytokines and other cell communication moleculeshave been described, and many of these molecules are expressed with inmore or less tissue- or cell-type restricted fashion. Thus, when it isdesired to target a subgroup of T cells, the CD4 T cell surface moleculemay be used for producing the conjugate of the invention. CD4-bindingmolecules are provided by the HIV virus, whose surface antigen gp42 iscapable of specifically binding to the CD4 molecule. Anapoptosis-enhancing Cari, mutant or peptide of the invention may beadvantageously targeted to T cells in the treatment of patient whosuffer from autoimmune reactions based upon T cells, such as lupuserythematodes patients.

The polypeptides, peptides and antisense sequences of the invention maybe introduced into cells by the use of a viral vector. The use ofvaccinia vector for this purpose is detailed in the Chapter 16 ofAusubel (1978-1995, 1999 and 2003). The use of adenovirus vectors hasbeen described, e.g., by Teoh et al (1998), Narumi et al (1998),Pederson et al (1998), Guang-Lin et al (1998), and references therein,Nishida et al (1998), Schwarzenberger et al (1998), and Cao et al(1998). Retroviral transfer of antisense sequences has been described byDaniel et al (1998).

In order to treat and/or prevent diseases in which Cari is involved, agene therapy vector comprising the sequence of an inhibitor of Cari, ifinhibition of apoptosis is required, or alternatively comprising thesequence of Cari if enhancement of apoptosis is required, for eitherinhibition or induction of Cari production and/or action respectively,may be injected directly into the diseased joint, for example, thusavoiding problems involved in systemic administration of gene therapyvectors, like dilution of the vectors, reaching and targeting of thetarget cells or tissues, and of side effects.

When using viruses as vectors, the viral surface proteins are generallyused to target the virus. As many viruses, such as the above adenovirus,are rather unspecific in their cellular tropism, it may be desirable toimpart further specificity by using a cell-type or tissue-specificpromoter. Griscelli et al (1998) teach the use of the ventricle-specificcardiac myosin light chain 2 promoter for heart-specific targeting of agene whose transfer is mediated by adenovirus.

Alternatively, the viral vector may be engineered to express anadditional protein on its surface, or the surface protein of the viralvector may be changed to incorporate a desired peptide sequence. Theviral vector may thus be engineered to express one or more additionalepitopes, which may be used to target, said viral vector. For instance,cytokine epitopes, MHC class II-binding peptides, or epitopes derivedfrom homing molecules may be used to target the viral vector inaccordance with the teaching of the invention.

Thus, Cari can be used for gene therapy by reducing or increase theendogenous amount of Cari at a desired site in a human patient.

The interaction of Cari with caspase-8 has several possibleconsequences:

The first is modulation of apoptosis. This is demonstrated herein in anin vivo assay wherein overexpressed Cari potentiates apoptosis inducedby overexpression of p55TNFR, P72 overexpression or by stimulation withFas-ligand. In one embodiment, the non-cleavable mutant p72 D600E wasshown to be more potent in Fas-ligand cell death potentiation than thewild type Cari. One possible explanation for this result may be thatCari is involved in the conversion of pro-caspase-8 into activecaspase-8. Therefore a non-cleavable Cari will continuously induceconversion of pro caspase-8 into active caspase-8 and increase apoptosisunlike the wild type Cari, which can be progressively cleaved andinactivated by active caspase-8. Cari has RNA binding motifs; therefore,its mechanism of action may involve changes in translation rate and/orturnover of different mRNA transcripts, which consequently effect theexpression of key proteins involved in modulation of cell death.

Secondly, the activity of Cari may be modulated. This is demonstratedherein by the ability of caspase-8 to cleave Cari. It is likely thatCari is inactivated by the cleavage. However, it is also possible thatthe activity of the Cari is changed, that novel activities are induced,or that the Cari polypeptide is activated by cleavage, just as thecaspases themselves.

Consequently, Cari, mutants, preferably the Cari/p72 D600E mutant, thepeptides, for example the caspase-8 binding domain in Cari comprisingamino acid residues from 414 to 437 (SEQ ID NO:4) and from residues 422to 437 (SEQ ID NO:5), oligonucleotides such as Cari antisense andspecific antibodies for Cari are useful in modulating the activity ofcaspase-8 and apoptosis.

Down-regulation of caspase-8 is desirable in situations where excessivecell death by apoptosis occurs. For instance, in inflammatory diseasessuch as multiple sclerosis with primary oligodendrogliopathy, autoimmuneuveoretinitis, diabetes, lupus, autoimmune myocarditis I, acute liverfailure regardless of etiology, HCV-mediated chronic hepatitis, chronicgastritis, e.g., type A gastritis, mixed connective tissue disease,(MCTD), Crohn's disease, and ulcerative colitis, it has been suggestedthat destruction of body tissue is caused by apoptotic signals.Therefore, it may be beneficial to patients suffering from thesediseases to down-modulate caspase-8 activity in those cells that aredestroyed by apoptotic cell death.

Similarly, the peptides or polypeptides of the invention such as Cari414-437 and Cari 422-437 may be targeted to other cell type involved inother diseases listed above and other diseases where an excess ofapoptotic cell death has been shown to mediate the damage in body tissueobserved.

Up-regulation of caspase-8 activity and increase of apoptosis by Carimay be employed in situations where excessive cell death is required.

For instance, in the above oligodendropathy, it is desired to inhibitcaspase-8 activity, in oligodendrocytes. The cell surfaceG-protein-coupled phospholipid lysophosphatidic acid receptor isexpressed in oligodendrocytes and in various other brain cells, but notin other tissues of the body. Since it has been demonstrated in one ofthe embodiments that TNF receptor signaling pathway or caspase-8dependent apoptosis requires the activity of CARI a small peptide ofCari, for example, the Cari polypeptide of 24 amino acid (Cari 414-437),which was found to bind caspase-8, can be targeted to theoligodendrocytes to inhibit apoptosis mediated by caspase-8. This may beachieved by either coupling said peptide or polypeptide to phospholipidlysophosphatidic acid, or by introducing the sequence of an antibodythat specifically recognizes said phospholipid lysophosphatidic acidreceptor into a viral vector, so that said viral vector specificallybinds to said phospholipid lysophosphatidic acid receptor.

Also, the antisense RNA, antisense oligonucleotide with sequencesderived from human Cari cDNA's, such as, AAGAGGATAAGGTAGAGCTCC(1169-1190) (SEQ ID NO:6) and/or from 3′-non-translated regionAATGACCAACCGTCCCTGGAC (3′ 26-47 bp) (SEQ ID NO:7), and ribozyme of theinvention may be targeted similarly to the above oligodendrocytes, orcorresponding cells in other diseases. In that case, the expression ofCari polypeptide is inhibited, rather than the expression of caspase-8itself. Inhibiting the expression of Cari may decrease the apoptoticeffect of caspase-8. However, decreasing the expression of Caripolypeptides may actually increase the effect of caspase-8, as certainendogenous Cari polypeptides are capable of acting as a negativeregulator of caspase-8 activity. The effect of using antisenseoligonucleotides and antisense RNA, and of ribozymes must therefore befirst tested, e.g., in the above-described assay, before such agents areconsidered for treatment.

On the other hand, there are certain situations where it may be desiredto increase caspase-8 activity. This may be the case in the same diseaseas noted above, e.g., in systemic lupus erythematodes. However, the celltypes that are to be targeted are different. For instance, in Lupus, theT cell population may contain autoreactive cells that are not destroyedin the thymus. Therefore, the caspase-8 up-regulating agent of theinvention should be targeted to T cells. It is preferable to target thecaspase-8 up-regulating agent to autoreactive cells. In some diseases,such as multiple sclerosis, certain T cell clones are presumed to play acritical role in development of the disease. The caspase8 up-regulatingagent according to the invention may therefore be targeted to suchcells, by using one or more antibodies specifically directed at thevariable region of the T cell receptor of the autoreactive T cellclones, for targeting the caspase-8 up-modulating agent of theinvention, which may be a Cari polypeptide, mutants or a peptideaccording to the invention.

Increasing caspase-8 activity and apoptosis by Cari can be used also fortreating cancer.

The present invention encompasses pharmaceutical compositions comprisingan active substance selected from one or more of a Cari polypeptide,mutants, preferably p72 D600E, a peptide such as the one comprisingamino acid residues from 414 to 437 (SEQ ID NO:4) and amino acidresidues from 422 to 437 (SEQ ID NO:5), vectors encoding such Cari,muteins thereof and fragments an antibody specific for Cari, a ribozyme,antisense RNA, or antisense oligonucleotide according to the invention.

The therapeutically effective amounts of the active protein(s) will be afunction of many variables, e.g., the route of administration, theclinical condition of the patient.

A “therapeutically effective amount” is such that when administered,Cari or its antagonist, exhibit biological activity. The dosageadministered, as single or multiple doses, to an individual will varydepending upon a variety of factors, including pharmacokineticproperties, the route of administration, patient conditions andcharacteristics (sex, age, body weight, health, size), extent ofsymptoms, concurrent treatments, frequency of treatment and the effectdesired. Adjustment and manipulation of established dosage ranges arewell within the ability of those skilled in the art, as well as in vitroand in vivo methods of determining the effect in an individual.

The invention further encompasses pharmaceutical compositions comprisinga viral vector capable of infecting mammalian cells wherein said vectorcomprises an operably linked promoter and a DNA sequence of theinvention coding for a Cari, mutants (e.g., p72 D600E), peptide (e.g.,Cari 414-437 or Cari 422-437), a ribozyme, an antisense RNA, anantisense oligonucleotide, or a Cari antibody according to theinvention. The viral vector may optionally comprise a coding sequenceoperably linked to a promoter, which encodes a peptide, or proteinlocated on the virus surface and which is capable of binding a surfaceprotein of a mammalian cell. The surface protein is preferably a proteinthat enables uptake of the viral vector, and is preferably expressed ina tissue- or cell-type specific manner, so as to enable targeting of theviral vector.

Cari, its muteins, fragments or derivatives the p72 D600E mutant,peptides Cari 414-437 or Cari 422-437, a ribozyme, an antisense RNA, anantisense oligonucleotide, or a Cari antibody according to the inventionmay also be used to isolate, identify and clone other polypeptides ofthe same class, i.e., those binding to caspase-8 or to isolatefunctionally related proteases or proteins, involved in theintracellular signaling process.

For this purpose the above immunoprecipitation system may be used, orthere may be used a developed system employing non-stringent Southernhybridization followed by PCR cloning (Wilks et al, 1989). In the Wilkset al publication, there is described the identification and cloning oftwo putative protein-tyrosine kinases by application of non-stringentsouthern hybridization followed by cloning by PCR based on the knownsequence of the kinase motif, a conceived kinase sequence. This approachmay be used, in accordance with the present invention using the sequenceof the Cari polypeptide to identify and clone those of related Caripolypeptides.

Another approach to utilizing the Cari polypeptide, muteins, fragmentsor derivatives thereof or Cari antibodies of the invention is to usethem in methods of affinity chromatography to isolate and identify otherpolypeptides or factors to which they are capable of binding, e.g.,other polypeptides or factors involved in the intracellular signalingprocess. Cari, its muteins, fragments or derivatives thereof, Cariantibodies may be individually attached to affinity chromatographymatrices and then brought into contact with cell extracts human fluidsor isolated polypeptides or factors suspected of being involved in theintracellular signaling process. Following the affinity chromatographyprocedure, the other polypeptides or factors which bind to the Caripolypeptide, or its muteins, fragments or derivatives thereof of theinvention, can be eluted, isolated and characterized.

The present invention also relates to the use of Cari and peptidesthereof such as the caspase-8 binding domain in Cari (residues 414-437or residues 422-437) or Cari antibodies for the preparation ofantagonist molecules to Cari, with potential therapeutic value. Thesemethods comprise the use of Cari and fragments thereof for the isolationof natural antagonists, small peptide antagonists and non-peptidechemical antagonists. High throughput screening uses robots to test thebinding activity of thousands of compounds (peptides or chemicalcompounds, e.g., created by combinatorial chemistry) against a Caritarget. The compounds tested may be obtained not only throughcombinatorial chemistry, but also from other high throughput synthesismethods. Automated techniques enable the rapid synthesis of libraries ofmolecules, large collections of discrete compounds, which can bescreened. Compounds may be also screening for inhibiting Cari or Cari(414-437) and caspase-8 interaction. Antagonistic molecules can be alsodetected by their capability of inhibiting binding of Cari topro-caspase-8, or inhibiting cell death mediated by Cari, caspase-8 orTNF receptor family ligands. Producing larger and more diverse compoundlibraries increases the likelihood of discovering a useful drug withinthe library.

As noted above, Cari its muteins, fragments or derivatives thereof, mayalso be used as immunogens (antigens) to produce specific antibodiesthereto. These antibodies may also be used for the purposes ofpurification of the Cari polypeptide either from cell extracts or fromthe medium of transformed cell lines producing Cari polypeptide, or itsmuteins or fragments. Further, these antibodies may be used fordiagnostic purposes for identifying disorders related to abnormalfunctioning of the caspase-8 mediated Fas or TNF system. Thus, shouldsuch disorders be related to a malfunctioning intracellular signalingsystem involving caspase-8, or a Cari polypeptide, such antibodies wouldserve as an important diagnostic tool. Such antibodies produced usingCari, especially fully humanized antibodies (e.g., intrabodies, seeabove), may have also therapeutic value.

It should also be noted that the isolation, identification andcharacterization of polypeptide according the invention or one of thesame class, might be performed using any of the well-known standardscreening procedures, for example, one of these screening procedures,the yeast two-hybrid procedure (see Stanger et al, 1995). Likewise, asnoted above and below, other procedures may be employed such as affinitychromatography, DNA hybridization procedures, etc. as are well known inthe art, to isolate, identify and characterize the polypeptide of theinvention or to isolate, identify and characterize additionalpolypeptides, factors, receptors, etc. which are capable of binding tothe polypeptides of the invention.

Having now described the invention, it will be more readily understoodby reference to the following examples that are provided by way ofillustration and are not intended to be limiting of the presentinvention.

EXAMPLES Example 1 Immunization of Mice for Generation of MonoclonalAntibodies Specific to Caspase-8

Following activation, caspase-8 is cleaved and assembled in two subunits (Sub-1 and Sub-2).

For the generation of antibodies specific to new possible epitopesformed following caspase-8 activation, synthetic peptides derived fromthe C-terminus of Sub-1 and N-terminus of Sub-1 and Sub-2 were used toimmunize mice.

The following peptides were used to immunize mice for the generation ofmonoclonal antibodies:

-   -   Peptide 179—The peptide CQGDNYQKGIPVETD (residues 360-374 of SEQ        ID NO:8) corresponding to the C-terminus of the large subunit of        caspase-8 (Sub-1), (epitope corresponding to residues        Cys360-Asp374 FIG. 1) was synthesized purified by reverse HPLC        and coupled to the carrier KLH through its natural cysteine, to        expose the peptide to the surface of the carrier.    -   Peptide 182—The peptide LSSPQTRYIPDEADC (SEQ ID NO:9)        corresponding to the N-terminus of the small subunit of the        caspase-8 (Sub-2, residues Leu385-Asp398) was synthesized        purified by reverse HPLC and coupled to carrier KLH through the        C which is not derived from the sequence of Sub-2.    -   Peptide 183—The peptide SESQTLDKVYQMKSKPRC (SEQ ID NO:10)        corresponding to the N-terminus of Sub-1 (residues        Ser217-Arg233), was synthesized purified by reverse HPLC and        coupled to carrier KLH through the C that is not derived from        the sequence of Sub-1.

Four immunizations and two boosts with the same amount of antigen(peptide-KLH) were administered to mice as follows:

-   -   First immunization with 50 μg of peptide-KLH were dissolved in        50 μl PBS and homogenized with 50 μl complete Freund's Adjuvant        and injected into the footpad of each of five 7 week old Balb/C        female mice.    -   For the second immunization, carried out 2 weeks after the first        immunization, mice were intramuscularly boosted with the same        amount of the peptide in a 50% (v/v) solution of incomplete        Freund's adjuvant.    -   For the third immunization, carried out two weeks after the        second immunization, mice were injected intraperitoneal with 50        μg of peptide-KLH in 50 μl PBS.

Sera of the injected mice were tested 10 days after the second and thethird immunization.

-   -   The fourth immunization (carried only for peptides 182 and 183),        was performed a month latter in similar way as the third        immunization.

One month after the fourth immunization (or third immunization for micechallenged with peptide 179) two boosts were carried out (in a similarway as the third and fourth immunization) within two-day interval.

Four days latter the spleen and inguinal lymph nodes of the two miceexhibiting the highest specific immunoreactivity were taken for fusionwith myeloma cells (Eshhar, 1985).

Example 2 Immunization of Rabbits for Generation of PolyclonalAntibodies Specific to Caspase-8

Rabbits were immunized with 179-KLH and 183-KLH for the generation ofspecific polyclonal antibodies.

The first immunization was carried out with 100 μg of peptide-KLH whichwas dissolved in 50 μl PBS and homogenized with 50 μl complete Freund'sAdjuvant and injected subcutaneously. A second immunization was carriedout two weeks later with the same amount of peptide-KLH and injectedintramuscularly two weeks later with incomplete Freund's adjuvant. Thesetwo immunizations were followed by two boosts of the same amount ofpeptide-KLH dissolved in PBS and administered subcutaneously at twoweeks interval.

Example 3 Hybridoma Preparation, Selection of Antibody Producing Clonesand Purification of Antibodies from Ascites Fluids

The fusion process and hybridoma cell selection were performed accordingto the protocols in Eshhar (1985). Briefly, a mixture of spleen andlymph node cells from 2 reactive mice 110×10⁶ were fused with 32×10⁶NSO/1 myeloma variant myeloma cells by a short incubation with PEG. ThePEG was first slowly diluted with DMEM and then completely removed bycentrifugation. The cells were re-suspended in DMEM-HAT medium,distributed in 96 wells plates at a concentration of about 2.5×10⁴cells/well and incubated in an 8% CO₂ incubator at 37° C. The medium inall the hybridoma wells was changed to DMEM supplemented with 10% HorseSerum (HS). Hybridoma culture supernatant samples were screened for thepresence of specific mAbs two weeks after the fusion by ELISA (describedin Example 12 below). Cells from wells, in which the presence ofspecific antibodies was detected in the culture supernatant, weretransferred to 24 well plates. Positive cells were subcloned twice; atthis stage all the sub-clones were found to be positive. The clones wereexpanded in 24 wells and then to 25 cm² T-flasks. The expanded cultureswere monitored for secretion of specific mAbs. Ampoules of cells frompositive cultures were frozen and stored in liquid nitrogen.

Out of approximately 700 clones screened for detecting specificantibodies to peptide 179 only one positive clone was found (mAb 179),out of 700 clones screened for detecting specific antibodies to peptide182 only 1 positive clone was found (mAb 182) and out of 1100 clonesscreened for detecting specific antibodies to peptide 183 only 2positive clones were found (mAbs 183.1 and 183.2). The positive cloneswere sub-cloned by limiting dilution in 96 well plates. Supernatantsfrom the growing clones were tested several times for specificantibodies by ELISA (described in Example 12).

Positive hybridoma clones were grown in tissue culture flasks in DMEMcontaining 15% horse serum and ampoules were frozen from part of thecultures. In parallel, cells of different hybridoma clones wereinjected, to 2-4 mice each, to obtain ascites fluids. The antibodieswere purified from ascites fluid by affinity purification using affigelbeads (affigel 15 Biorad) cross-linked with BSA (Pierce Cat 77116)coupled to the synthetic peptide used for mice immunization (peptides179, 182 or 183).

For antibody purification, ascites precipitated by 50% ammonium sulfatewas dialyzed against PBS for 16 hours at 0° C. Following dialysis,aliquots were incubated with 1 ml affigel-BSA-peptide beads for 16 hoursat 0° C. and the pre-incubated beads were used to pack a 1 ml column.Initially the column was washed with 10 ml PBS, followed by a wash with10 mM Tris pH 7.5 containing 1 M NaCl and a wash with PBS. Theantibodies were eluted from the column with a solution containing 100 mMglycine HCl, pH 2.7 and 0.5M NaCl. 1 ml fractions were collected intubes containing 40 μl Tris base for the neutralization of the eluent.From 25 ml ascites about 5-13.6 mg-purified antibodies was obtained.

Example 4 Monoclonal Antibodies Isotype

The isotype of monoclonal antibodies was determined using a commercialisotyping kit (Southern Biotechnology Associates, INC cat 5300-05)according to the manufacturer's assay procedure. mAbs 183 and 179 wereidentified as IgG1, whereas mAb 182 was found to be of the IgM class.

Example 5 Immunoprecipitation of Caspase-8 with mAbs 179, 182 and 183

The different monoclonal anti-caspase-8 antibodies described in theExample 3 above were tested for their capacity to immunoprecipitatecaspase-8 (see Example 13 below) from lysates of resting and activatedBJAB cells. BJAB line is a continuous lymphoma cell line derived fromthe African case of Burkitt's lymphoma (Clements et al, 1975). BJABcells were stimulated with Fas-ligand for one hour. Cell lysates wereprepared from BJAB cells before and after stimulation. Followingimmunoprecipitation with mAbs 179, 182 and 183 (as described in Example13) the “depleted lysate” and the caspase-8 eluted with thecorresponding peptides were analyzed by SDS-PAGE and Silver staining orby Western blot analysis using anti Sub-1 antibody as the first antibody(Cell Signaling Technology Caspase-8 ICI2 Cat 9746).

FIG. 2 shows a Western blot analysis (performed as described in Example11 below) of total cell extracts and “depleted lysates”, obtained afterimmunoprecipitation with mabs 179, 183.1 and 183.2 and 182.

In non-stimulated cells (lanes 2, 4, 6, 8 and 10), a band doubletcorresponding to pro-caspase-8 isoform α1 and α2 (pro-caspase-8 53/55kDa) was detected in total cell extracts (lane 2) and in depletedlysates obtained with anti-183 and anti-182 antibodies (lanes 6, 8 and10) in contrast no pro-caspase-8 was detected in depleted lysatesobtained with mAb 179 (lane 4). These results indicate that mAb 179immunoprecipitates pro-caspase-8.

In stimulated cells the levels of pro-caspase-8 in total cell extractwere lower (lane 1). Additional smaller bands corresponding to activatedcaspase-8 fragments appeared upon activation i.e. a doublet of partiallyprocessed caspase-8 corresponding to isoform α1 and α2 (partiallyprocessed caspase-8 p 41/43, lacking Sub-2) and a smaller bandcorresponding to Sub-1 (p 20). Depletion of the minute amounts ofpro-caspase-8 and activated caspase-8 fragments by the mabs was testedon lysates of Fas-ligand stimulated cells (FIG. 2 lanes 3, 5, 7, 9 and11).

It should be noted that depletion of Sub-1 by mAb 182, specific toSub-2, was also tested since activated caspase-8 comprises Sub-1 boundto Sub-2 and therefore removal of Sub-2 by immunoprecipitation with mAb182 should consequently lead to depletion of Sub-1.

Immunoprecipitation of caspase-8 from stimulated cell lysates show thatmAbs 182, 183.1 and 183.2, similar to the normal mouse serum control(FIG. 2 lane 11), did not remove the small amounts of remainingpro-caspase-8 or the active caspase-8 fragments (lanes 9, 7, 5 and 11respectively). In contrast to these results, treatment of the celllysates with mAb 179 (lane 3), efficiently removed all thepro-caspase-8, as well as the active caspase-8 fragments.

FIGS. 3 a (Western blot analysis) and 3b (protein detection by Silverstaining) show that immunoprecipitated pro-caspase-8 and activecaspase-8 fragments by mAbs 179, 182 and 183.1 and 183.2 antibodiescould be efficiently recovered into the supernatant by competition withthe respective peptides against which the various antibodies were beenraised (Example 13).

In non-stimulated cells (FIG. 3 a, lanes 2, 4, 6, 8 and 10 and FIG. 3 b,lanes 2, 3, 6, 8, and 10), pro-caspase-8 is efficiently recovered byimmunoprecipitation with mAb 179 and competition with peptide 179 (FIGS.3 a and 3 b, lane 8). In stimulated cells, in spite of the small amountof pro-caspase-8 left after activation, immunoprecipitation with mAb 179resulted in effective recovery of the protein (FIGS. 3 a and 3 b, lane9). Some recovery of pro-caspase-8 could be observed in non activatedcells by mAb 183.2 (FIG. 3 a, lane 6) and in activated cells by mAb183.1 (FIG. 3 a, lane 5) where active fragments of caspase-8 could berecovered in lysates of activated cells by mAbs 182 (FIG. 3 a, lane 3),183.1 (FIG. 3 a, lane 5) and 183.2 (FIG. 3 a, lane 7, only p20).

The results obtained indicated that the mAb 179 developed against thepeptide corresponding to the C-terminus of Sub-1 (179 epitope) is veryefficient for immunoprecipitation and purification of pro-caspase-8,even present in trace amounts, as well as for activated caspase-8.

Polyclonal antibody specific to the same 179 epitope (prepared asdescribed in Example 1 above) was generated to investigate whether the179 epitope has the unique capability of eliciting antibodies, which canbe generally used for the efficient immunoprecipitation and purificationof pro-caspase-8 and active caspase-8. The “depleted lysates” obtainedby immunoprecipitation with polyclonal antibody specific to epitope 179(lanes 5 and 6 for activated and non-activated cells, respectively) orby monoclonal antibody specific to epitope 182 (lanes 7 and 8 foractivated and non-activated cells, respectively) were compared. Theresults in FIG. 4 clearly show that indeed, pro-caspase-8 and caspase-8fragments from stimulated cell lysates are also efficiently removed fromthe cell lysate with polyclonal anti 179 antibodies and even moreefficient than with monoclonal anti182 antibody.

In parallel immunoprecipitation and recovery of pro-caspase-8 fromresting cells lysates carried out with mAb 183 and polyclonal antibodyspecific to the 183 epitope (described in Example 1) were compared tothose obtained with mAb 179. FIG. 5 shows that immunoprecipitation ofpro-caspase-8 by mAb 183 and poly 183 is ineffective whileimmunoprecipitation of pro-caspase-8 by mAb 179 is remarkably superior.

An additional caspase-8 derived fragment of about 5.6 kDa is observedonly in immunoprecipitates carried with mAb 179 (lane 3). Antibodiesdeveloped against the region of caspase-8 that corresponds to theC-terminus of the large caspase Sub-1 have a unique ability to impose onthe caspase a novel mode of processing.

The results observed above indicate that epitope 179 of caspase-8,unlike other epitopes, has the special capability of eliciting specificantibodies that are very efficient for immunoprecipitation ofpro-caspase-8 and activated caspase-8 and are able to inducepro-caspase-8 autoprocessing.

Example 6 Isolation and Identification of a Caspase-8 BindingPolypeptide (Cari)

Due to its capability to efficiently immunoprecipitate caspase-8, mAb179 was exploited to co-immunoprecipitate caspase-8 and caspase-8-boundproteins.

BJAB cells (Steinitz et al, 1975) were stimulated with Fas-ligand forone hour and cell lysates were prepared from cells before and afterstimulation. Following immunoprecipitation and elution, as described inExample 13, the recovered proteins were resolved by SDS-PAGE anddetected by Silver-staining. Immunoprecipitation with mouse IgG1 servedas the negative control. The results in FIG. 6 show that a polypeptideof an apparent molecular weight of about 72.5 kDa (herein called p72) isco-precipitated with pro-caspase-8 (p 53/55) in lysates from restingcells (lane 3), but not with active caspase-8 in lysates from stimulatedcells (lane 4).

In addition, a p72 polypeptide was found to co-immunoprecipitate withpro-casapse-8 also in lysates prepared from non-stimulated HeLa, Raji,H9, K562, HL-60, CEM and Hut78 cells (ATCC).

These results suggest that a polypeptide, p72, is generally bound topro-caspase-8 but not to active caspase-8.

The band in the SDS-PAGE corresponding to p72 was excised, trypsindigested and subject to limited sequence analysis and to massspectroscopy analysis. Seven peptides obtained by trypsin digestion wereused to search a protein database deduced from nucleotide sequences (orESTs). The protein sequence matched part of a predicted protein sequenceof a human EST clone (SEQ ID NO:1) found in the gene bank (accessionnumber gi/2988397/gbAAC08052.1/(AC004475) whose function was unknown.

Example 7 Generation of the Full-Length cDNA Encoding p72

The full-length cDNA encoding p72 was generated as follows:

The EST from (Example 6) was used to screen a TIGR Human gene index andthe THC report (THC510568 SEQ ID NO:1) containing the consensus of allthe ESTs that fit this sequence was obtained.

A DNA clone encoding part of the predicted polypeptide was purchasedfrom Incyte Genomics (IMAGE #2964545). The clone lacked the nucleotidesequences encoding the first methionine and the 6 succeeding amino acids(i.e., 21 nucleotides). The mouse and human sequences of these proteinswere found to be highly similar (about 90% identity), thus thenucleotide sequences encoding the first methionine and the 6 succeedingamino acids of the mouse protein which were not missing in the mouseESTs were compared to the working draft sequence of the human genome inorder to complete the missing human sequence. A hit was obtainedcorresponding to the sequence of Homo sapiens chromosome 19, cloneLLNLR-232E12. This clone confirmed the nucleotide sequence, whichencodes the missing 7 amino acids of p72. The full-length cDNA of p72was obtained by two PCR rounds (Takara ExTaq, Takara, cat # R001A wasused), which are schematically represented in FIG. 8.

In the first PCR the clone obtained from Incyte Genomics was used as thetemplate with the forward primer: CTCAAGATGGACAACCGGGATGTTGCAGGAAAGG(SEQ ID NO:11) synthesized to contain 15 (underlined) out of the 21missing nucleotides together with the existent sequence of p72 (FIG. 8,primer 2), and the reverse primer: CCACTCGAGTCAGTAGTAAGGCCGTCTGGGATT(SEQ ID NO:12) containing the 3′ region ending with the stop codon (FIG.8, primer 3).

The second PCR comprises as the template the PCR product of the firstPCR round and the forward primer: AATGGATCCATGAGTCTCAAGATGGACAACCGGGA(SEQ ID NO:13) containing the whole 21 missing nucleotides and 5existent nucleotides (FIG. 8, primer 1) and the same reverse primer(FIG. 8, primer 3). The whole cDNA encoding p72 was recovered andsequenced (SEQ ID NO:2), and the amino acid sequence was predicted fromthe nucleotide sequence (SEQ ID NO:3).

Comparison of the sequence obtained in the THC report (THC510568 SEQ IDNO:1) containing the consensus of all the ESTs, and the polypeptidepredicted by the generated full length cDNA (SEQ ID NO:3) in FIG. 13shows the missing 7 first amino acids in the ESTs the missing 25 aminoacids sequence in the ESTs and inaccuracy of amino acid 397 (prolineinstead of leucine).

P72 polypeptide was found to contain three conserved motifs (FIG. 7):the C motif a coiled motif, two tandem located ‘SURP’ (also called‘SWAP’ motifs, denoted as S FIG. 7) (Denhez and Lafyatis, 1994) close tothe N terminus of the polypeptide, and one C terminally located‘G-patch’ (FIG. 7 denoted as G) (Aravind and Koonin, 1999). Both theSURP and the G-patch motifs are believed to contribute to RNA-binding,suggesting that the target of p72 may be a RNA molecule. Thus p72 wasrenamed to Cari (Caspase-8 Associated polypeptide with RNA bindingmotifs)

Example 8 Cleavage of Cari by Caspase-8

As shown in Example 6, Cari is bound only to pro-caspase-8 and not toactive caspase-8 as tested one hour after stimulation. Somepro-caspase-8 can be still detected after 20 minutes stimulation. Todetermine whether Cari can be co-precipitated with pro-caspase-8 atshorter stimulation times, BJAB cells activated for only 20 minutes werelysed and immunoprecipitated with mAb 179. Following immunoprecipitationand elution, caspase-8 and bound polypeptides were resolved by SDS-PAGEand the polypeptides were detected by Silver staining. One band of 72.5kDa (FIG. 9, lane 3) probably corresponding to Cari wasimmunoprecipitated in lysates from cells before stimulation while after20 minutes stimulation, in addition to Cari, a polypeptide with a lowerapparent molecular weight of about 68 kDa was detected (FIG. 9, lane 4).Both polypeptides, the 72.5 and 68 kDa, immunoprecipitated from BJABcells, were subjected to mass spectroscopy analysis. Aftertripsynization, both polypeptides exhibited similar peptide profileexcept one clear difference, an additional peptide of sequenceFRPNPLNNPR (residues 632-641 of SEQ ID NO:3) was present in the 72.5 kDa(Cari) at the C-terminus but absent in the 68 kDa polypeptide.

This result suggests that upon cell stimulation a fragment of about 4.5kDa is removed from the C-terminus of Cari, probably by activatedcaspase-8, resulting in a smaller polypeptide with an apparent molecularweight of 68 kDa, which is still bound to the remaining pro-caspase-8.

It is conceivable that residue D 600 located at the C-terminus of Cari(FIG. 7) could be a candidate residue for cleavage, because the putativefragments resulting from such a cleavage exhibit similar molecularweight as the Cari fragments detected in vivo following 20 minutesstimulation.

In order to test whether, as suggested, Cari is a substrate of caspase-8and D 600 is the target residue for cleavage, an in vitrotranscripted-translated and radioisotope labeled (S³⁵) Cari (TnT system)was subjected to the action of recombinant active caspase-8. Cari cDNAwas expressed in vitro in reticulocyte lysates in the presence of ³⁵Smethionine using the TnT T7 Coupled Reticulocyte Lysate System, andsubjected to cleavage by recombinant active caspase-8 (each Sub-unit 1and 2 prepared separately in E. coli mixed and re-folded together invitro). Briefly, in-vitro synthesized ³⁵S labeled Cari was incubated for30 min. in protease buffer (25 mM HEPES, pH 7.5, 0.1% CHAPS, 5 mM EDTAand 2 mM DTT) at 37° C. in the presence or the absence of bacteriallyproduced active caspase-8. Proteins and their fragments were separatedon SDS-PAGE and the results visualized by phospho-imaging. The results(FIG. 10) show that in the absence of caspase-8 only the 72.5 bandcorresponding to full length Cari (lane 1) was detected. This banddisappears after addition of activated caspase-8 for 1 hour and a newsmaller fragment corresponding to 68 kDa appears (lane 4). This resultindicates that the protein encoded by the Cari cDNA used as substrate,is effectively cleaved by caspase-8.

In addition, the TnT transcription translation system was used also toproduce in vitro two different Cari mutants: (1) Cari in which theresidue D 600, suspected from the in vivo experiments to be the targetresidue for caspase-8, was mutated to E (D600E), and (2) a deleted Carimissing the residues down-stream D600 (i.e., the expressed protein willexhibit the 1-600 residues).

Cleavage of the above two Cari mutants was tested in the presence (FIG.10, lanes 5 and 6, respectively) or in the absence (lanes 2 and 3,respectively) of active recombinant caspase-8. As shown in FIG. 10(lanes 3 and 6 respectively), the same protein profile of Cari D600Emutant is observed in the presence or the absence of caspase-8,indicating that caspase-8 does not cleave the Cari D600E mutant. TheCari 1-600 mutant co-migrates with the 68 kDa fragment produced aftercleavage of the wild type Cari and is not further cleaved by addition ofcaspase-8 (lanes 2 and 5). These results show that, upon activation,caspase-8 cleaves Cari at the D600 residue.

Studies carried out in vivo suggest that cleavage of Cari by caspase-8occurs rapidly in cells, within 5-20 minutes after Fas ligandstimulation (FIG. 11), and that the cleaved Cari (or rather—its largerfragment) may remain associated with pro-caspase-8.

Example 9 Functional Characterization of Cari

To analyze the effect of Cari on apoptotic cell death induced by the TNFreceptor signaling pathway, Cari cDNA or antisense Cari (a/s), or avector without p72 cDNA insert as the negative control was used (pc).The cDNA was inserted into the pcDNA 3.1 expression vector (availablefrom Invitrogen) and co-transfected with p55 TNFR receptor DNA insertedinto the pcDNA3 vector (Invitrogen) and with the reporter gene greenfluorescence protein (GFP) inserted into the pEGFPC1 expression vector(Clontech), into HEK 293 cells constitutively expressing the T antigen.

After 24 hours the transfected cells were examined under a fluorescentmicroscope and cell death was scored by determining the number of cellsdisplaying apoptotic morphology out of the total population offluorescent cells. Overexpression of Cari was found to potentiate thecell death induced by overexpression of the p55 TNF receptor (FIG. 12 a,p72). In contrast, when antisense cDNA construct was used forco-transfection (FIG. 12 a p72/a/s), the cells were protected from deathinduced by overexpression of the p55 TNF.

This result indicates that Cari modulates apoptotic cell death inducedby the TNF receptor-signaling pathway.

In general, triggering of a receptor like CD120a by FasL requires theaddition of a protein synthesis inhibitor like cycloheximide in order toachieve a strong signal for apoptosis. To analyze the effect of Cari oncell death induced by the Fas signaling pathway, the effect of Carioverexpression on Fas ligand mediated cell death, without addition ofcycloheximide, was monitored in HEK 293 cells constitutively expressingthe T antigen (HEK-293 T). In the experiment, HEK-293 T cells were cotransfected with a vector pcDNA3.1 or pcDNA3.1 encoding Cari or Cariantisense (pc, p72 and p72 a/s respectively) and a vector pSBC-2(Dirkset al, 1983) encoding the reporter gene SEAP (secreted alkalinephosphatase). After 24 hours, the transfected cells were induced withFas-ligand for 16 hours and the growth medium was replaced with freshgrowth medium. Cell death was calculated by determining the amount ofSEAP secreted into the growth medium during 24 hours (detected byenzymatic reaction as described by Boldin et al). The results in FIG. 12b indicate that overexpression of Cari, but not Cari antisense, causeddeath in combination with Fas-ligand stimulation. The results show alsothat overexpression of Cari in the absence of Fas-ligand stimulationdoes not have any effect on cell death (not shown).

The effect of pSuper-Cari overexpression, a system for stable expressionof short interfering Cari RNAs, which supposedly will blockage Cari, wastested on apoptosis induced in HeLa cells by overexpression of Mach a1(caspase-8) or by a chimera comprising the extracellular part of p55TNFR1 fused to the transmembrane and intracellular part of FAS receptor(CI*).

HeLa cells (2×10⁵ cells) were seeded and cotransfected using 2 μg Macha1 or CI* DNA containing plasmid (backbone plasmid, pcDNA3 fromInvitrogen) and 3 μg psuper vector (Brummelkamp et al, 2002) orpSuper-Cari (pSuper-Cari is a 1:1 mix of psuper plasmids containingsequences derived from human Cari cDNA's AAGAGGATAAGGTAGAGCTCC(1169-1190 SEQ ID NO:6) or from 3′-non-translated regionAATGACCAACCGTCCCTGGAC (3′ 26-47 bp SEQ ID NO:7) and 0.5 μg reporter GFPgene containing plasmid (backbone plasmid, pRGFP Clontech). 27 hourspost transfection, the cells were examined under a fluorescentmicroscope and cell death was estimated by the morphology of the GFPcontaining cells.

The results are summarized in FIG. 16. While death of cells is inducedby overexpressing Mach a1 or Cl* (apoptosis is more elevated in Mach a1than in CL* overexpressing cells), cells transfected with pSuper-Caritogether with Mach a1 or Cl* show significantly reduced or no apoptosis.These results demonstrate that induction of apoptosis through the TNFreceptor signaling pathway or by caspase-8 requires the activity ofCari.

Example 10 Western Blot Analysis for Detection of Caspase-8Immunoreactive Serum

A mixture of recombinant purified Sub-1 and Sub-2 was used for Westernblot analysis of antibodies developed to synthetic peptides. Briefly a12% SDS Poly Acryl amide gel was loaded with 100 ng/lane of a mixture ofSub-1 and Sub-2 under reducing conditions (40 mM DTT). One lane wasloaded with Low Molecular Weight Markers (LMW). The proteins separatedon the gels were transferred by electro elution to PVDF high bond-P(Amersham) membranes. The membranes were incubated in PBS containing 5%low-fat milk, 0.05% Tween 20, for 16 hours. The membranes were cut intostrips and each strip was incubated for 1 hour at room temperature withthe mouse antiserum (diluted 1/2000). Membrane strips were washed withPBS containing 0.05% Tween 20 (3×15 min) and incubated for one hour withthe second antibody—goat anti-mouse conjugated to horseradish peroxidase(diluted 1:10.000, Jakson) for 1 hour at room temperature.

The strips were washed with PBS containing 0.1% Tween 20 (3×15 min). Thepositive bands were detected by enhanced chemiluminescence (ECL,Amersham).

For Western blots performed in Example 5 antibodies specific to Sub-1were used (Cell Signaling Technology Caspase-8 ICI2 Cat 9746).

Example 11 ELISA for Hybridoma Clones Screening

The direct ELISA for screening hybridoma producing specific antibody wasperformed as following: 96 wells plates were coated with 50 μl/well ofBSA-peptide (or BSA alone for control plates) at a concentration of 2.5μg/ml in binding solution (0.1 M Na₂HPO₄, pH 9) for 1 hour at 37° C. or16 hours at 4° C. Subsequently the plates were washed 3 times with PBS-T(PBS with 0.05% of Tween-20) and loaded with 200 μl/well of blockingsolution (1% hemoglobin in PBS) for 1 hour at 37° C. and washed 3 timeswith PBS. 50 μl of hybridoma culture supernatant or diluted standards(with PBS-T) were loaded per well and incubated for 1 hour at 37° C. or4 hours at 22° C. After this incubation period the wells were washed 6times with PBS-T. A second antibody, anti mouse antibody conjugated toHRP (Jackson 115-025-100) was diluted 1:5000 in PBS-T, incubated for 1hour at 37° C. and washed away by washing 6 times with PBS-T. Thesubstrate for HRP was freshly prepared (2.2 ml of 0.2M Na₂HPO₄, pH 9.2,1.4 ml of 0.2 M citric acid, pH 4.35, 6.4 ml H₂O, 10 mg ABTS and 1 μlH₂O₂) and 50 μl/well were loaded and incubated at 22° C. until colordeveloped (about 5-80 minutes). The color reaction was stopped by adding50 μl/well 0.2 M citric acid. The plates were read at 405 nm.

As a positive control antibody, positive mouse antiserum diluted 1:1000was used and as negative control media.

Example 12 Immunoprecipitation of Caspase-8

For every immunoprecipitation 10⁸ cells were used. Cells were collectedand lysed by incubation in 1% NP-40 lysis buffer and complete proteaseinhibitor (complete protease inhibitor cocktail tablets from RocheMolecular Biochemicals) on 0° C. for 40 min. The cell lysates werealiquoted in Eppendorf tubes, centrifuged at 14000 rpm for 10 minutes at4° C. and the supernatant collected in a new tube. The cell lysates weresubjected to a pre-clearing step, intended to remove proteins that bindnon-specifically to the protein-G-sepharose. For pre-clearing, celllysate was pre-incubated with PBS pre-washed protein-G-sepharose(Pharmacia) and with mouse IgG for 2-3 hours at 4° C. Following thisincubation the lysates were centrifuged in Eppendorf tubes for 14000 rpmfor 30 seconds, the protein G-sepharose was discarded and thepre-cleared supernatant collected. Purified monoclonal antibody (ormouse IgG 1 kappa for negative controls) and PBS pre-washedprotein-G-sepharose were mixed and incubated with the pre-clearedsupernatant for 4- to 16 hours at 4° C. Following this incubation periodthe unbound material denoted “depleted lysate” was collected bycentrifugation (30 seconds at 14000 rpm) and the bound material waseluted by washing the sepharose beads 6 times with lysis buffer and byincubation with an “eluting solution” containing 0.2% NP-40 lysisbuffer, protease inhibitors and 400 μg/ml peptide used for immunization(300 μl eluting solution/100 μl sepharose) for 2 hours at 22° C. Thetubes were spun for 5 minutes at 5,000 rpm and the supernatant denoted“caspase-8 eluate” transferred into a new tube.

Example 13 Assessment of the Effect of the Non-Cleavable Mutant p72D600E in Fas-Mediated Apoptosis

Cari was found to be cleaved by active caspase-8 and the specificcleavage site was demonstrated by generating a non-cleavable mutant (p72D600E) (Example 8). Transient overexpression of Cari was shown topotentiate the apoptotic activity of Fas ligand-treated cells (Example8). To assess the effect of the non-cleavable mutant p72 D600E over thewild type polypeptide in Fas mediated apoptosis, BJAB cells weregenetically engineered to produce constitutively either the mutant orthe wild type Cari and the effect of Fas ligand in the engineered cellswas monitored. To generate the constitutive production, the cDNAsencoding each of the two Cari proteins were inserted in the pcDNA 3.1expression vector. Transfectant BJAB cells were selected in 1500 μg/mlneomycin and isolates were collected and tested for Cari production byWestern blot analysis of the cell lysates. Isolates producing similarlevels of Cari wild type and mutant were further selected. The survivalof BJAB cells was monitored after Fas ligand application to controlcells (B1), cells constitutively expressing transfected p72 (B2) andcells constitutively expressing transfected D600E p72 (B3). The resultsin FIG. 14 show that the cells expressing the non-cleavable Cari mutantare more efficiently killed by Fas ligand treatment than thoseexpressing the wild type Cari. One possible explanation for this resultis that Cari might be involved in the conversion of pro-caspase-8 intoactive caspase-8. Therefore a non-cleavable Cari will continuouslyinduce conversion of pro-caspase-8 into active caspase-8 resultingincrease of apoptosis, unlike the wild type p72, which will be cleavedand probably inactivated by active caspase-8.

To test the possible involvement of Cari in the activation of caspase-8,the rate of pro caspase-8 conversion into active caspase-8 upon Fasligand treatment in BJAB cells expressing wild type Cari or theuncleavable Cari mutant was monitored by western blot analysis withanti-caspase-8 specific antibodies for the detection of pro-caspase-8and activated caspase-8. The results obtained indicated that the rate ofcaspase-8 activation is higher in BJAB cells producing the mutant Cari(not shown).

These results suggest that p72 is involved in activation of caspase-8and that the increase in anti-apoptotic activity mediated by Fas ligandobtained with cells expressing the mutant p72 D600E over the wild typeprotein is due to the fact that the former protein remains active (isnot cleaved) in spite of the presence of active caspase-8.

Example 14 Determination of the Domain in Cari Responsible for BindingCaspase-8

Deletion studies were carried out with Cari to determine the minimalamino acid sequence in Cari responsible for binding caspase-8. Cari wasprogressively deleted and the resulting fragment was ligated to a GFPreporter gene and introduced into pcDNA3.1/HisC vector (Invitrogen). TheCari constructs (10 μg) were co-transfected into HeLa cell together witha vector (10 μg) containing pro-caspase-8 (Mach a1 (C360S) introduced inpcDNA 3 vector (Invitrogen).

24 hours post transfection, the cells were lysed in 1% NP-40 lysisbuffer. Caspase-8 was immunoprecipitated with antibody 179 and protein Gfor two hours. The precipitated complex was washed 5 times with washingbuffer (0.2% NP-40 buffer) and eluted in washing buffer containingpeptide 179. The immunoprecipitated proteins were resolved by SDS-PAGEand subjected to Western blot analysis using anti caspase-8 antibody1C12 (1:2000, Cell Signaling Technology) and polyclonal antibody againstE. coli produced His-Cari (1:2000).

FIG. 7 shows the schematic representation of Cari including theCoiled-coil motif, SURP-or SWAP motif, G-patch motif, the NLS motifs,the caspase-8 cleavage site D600 and the corresponding amino acidspanning the above motifs. Table II summarizes the results of binding ofCari full and deleted construct to caspase-8.

D600, the cleavage site of caspase-8 in Cari is probably not requiredfor its binding since the D600E Cari mutant does bind caspase-8. Thedeletion construct 393-645 which lacks part of the N-terminal of theprotein including some of the above Cari motifs (Coiled-coil motif,SURP-or SWAP motif, G-patch motif, the NLS motifs) but contains theintact C terminus of the protein still binds caspase-8, indicating thatunlike the C terminus end, those motifs are not required for bindingcaspase-8. The G-patch motif is probably not necessary as well, asdemonstrated with the deletion construct 393-437 which lacks the G-patchmotif and still binds to the caspase. The protein could be furtherdeleted from its N-terminal end to get a 24 amino acid peptideSVQDLKGLGYEKGKPVGLVGVTEL (construct 414-437) (SEQ ID NO:4), which stillbinds to caspase-8. Additional deletion of 16 amino acids to suchpeptide, from the N terminal end, resulted in a 9 amino acid peptide(construct 429-437) that failed to bind caspase-8. Thus, a deletion ofonly 7 amino acids from the N terminal of the 414-437 construct,resulting in a 16 amino acid peptide (construct 422-437), was tested andwas found to bind to caspase-8. Thus, the smallest amino acid able tobind caspase-8 was found to be a 16 amino acid peptide of sequenceGYEKGKPVGLVGVTEL (construct 422-437) (SEQ ID NO:5; FIG. 15).

TABLE II Binding to Cari Construct Caspase-8 Comment Full length Cari +(1-645) Mutant D600E + Non-cleavable at residue 600 (1-645)  1-260 −N-terminal end including only the first SURP module  1-330 − N-terminalend including two SURP modules  1-352 − N-terminal end including twoSURP modules and the first NLS  1-427 − N-terminal end including twoSURP modules and the first NLSs  1-490 + N-terminal end including twoSURP modules and all 3 NLSs  1-531 + N-terminal end including two SURPmodules and all 3 NLSs 179-645 + Including two SURP modules and theC-terminal end 260-645 + Including only the second SURP module and theC- terminal end 370-645 + Including only two NLSs and the C-terminal end370-645 + Similar to 370-645 mutant with mut NLS but with the NLSsequence mutated 393-645 + Comprising the C-terminal end without any NLS370-484 + Containing NLS and part of the C-terminal end excluding theG-patch motive 370-484 + Similar to 370-484 mutant with mut NLS but withthe NLS sequence mutated 438-484 − GFP-fusion to C-terminal end393-437 + GFP-fusion to C-terminal end 398-437 + GFP-fusion toC-terminal end 406-437 + GFP-fusion to C-terminal end 414-437 +GFP-fusion to C-terminal end 429-437 − GFP-fusion to C-terminal end422-437 + GFP-fusion to C-terminal end

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1. An isolated polypeptide capable of binding to pro-caspase-8 selectedfrom the group consisting of: a) a polypeptide consisting of the aminoacid sequence of SEQ ID NO: 3 (Cari); b) a variant of Cari (SEQ ID NO:3) comprising no more than ten amino acid changes and retains thecapability of binding to pro-caspase-8; c) a fragment of Cari (SEQ IDNO: 3) having the sequence of SEQ ID NO: 5 and includes thepro-caspase-8 binding site; and d) a derivative of Cari (SEQ ID NO: 3),which is prepared by modifying the —NH₂ group at the N-terminus or the—COOH group at the C-terminus or the functional groups that appear asside chains of one or more amino acids residues of the polypeptides of(a) to (c), without changing one amino acid to another one of the twentycommonly occurring natural amino acids, without the proviso that saidpolypeptide is not DF518-3.
 2. A polypeptide according to claim 1, beingencoded by SEQ ID NO:2.
 3. A polypeptide according to claim 1, which iscleaved in vitro by caspase-8.
 4. A polypeptide according to claim 1,which is cleaved in vivo by caspase-8.
 5. A polypeptide according toclaim 1, which is cleaved in vitro or in vivo by caspase-8.
 6. Apolypeptide according to claim 1, wherein said polypeptide is saidfragment of SEQ ID NO:3, or said derivative thereof, having adominant-negative effect in the activity of the endogenous Caripolypeptide.
 7. A polypeptide according to claim 6, capable ofinhibiting the apoptotic effect of caspase-8.
 8. A polypeptide accordingto claim 1, wherein said polypeptide is said fragment of SEQ ID NO: 3,or said derivative thereof, capable of inhibiting the interaction ofCari (SEQ ID NO: 3) and a caspase-8.
 9. A polypeptide according to claim8, consisting of the amino acid sequence in SEQ ID NO:4.
 10. Apolypeptide according to claim 8, consisting of the amino acid sequencein SEQ ID NO:5.
 11. A polypeptide according to claim 1, capable ofincreasing the apoptotic effect of caspase-8.
 12. A polypeptideaccording to claim 11, wherein a variant of Cari (SEQ ID NO: 3) is CariD600E mutant.
 13. An isolated DNA sequence encoding a polypeptideaccording to claim
 1. 14. A DNA sequence according to claim 13, encodingthe polypeptide of SEQ ID NO:3.
 15. A DNA sequence according to claim13, consisting of the DNA of SEQ ID NO:2.
 16. A DNA sequence accordingto claim 13, wherein the polypeptide is Cari D600E mutant.
 17. A DNAsequence according to claim 13, consisting of the sequence encoding SEQID NO:4.
 18. A DNA sequence according to claim 13, consisting of thesequence encoding SEQ ID NO:5.
 19. A vector comprising a DNA sequenceaccording to claim
 13. 20. A vector according to claim 19, wherein thevector is an expression vector.
 21. A vector according to claim 19,wherein the vector is a viral vector.
 22. A vector having a DNAregulatory sequence functional in cells for enabling endogenous geneactivation of an endogenous gene encoding the Cari polypeptide (SEQ IDNO:3), said vector further including targeting sequences correspondingto portions of said endogenous gene such that, after a step ofhomologous recombination, said regulating sequence will be appropriatelytargeted to allow said endogenous gene activation.
 23. An isolated hostcell comprising a vector according to claim
 19. 24. A method ofproducing a polypeptide capable of binding to pro-caspase-8, comprisinggrowing an isolated host cell according to claim 23, and isolating thepolypeptide produced.
 25. A method according to claim 24, wherein thecell is a eukaryotic cell.
 26. A method according to claim 25, whereinthe eukaryotic cell is a mammalian, insect, or yeast cell.
 27. A methodaccording to claim 26, wherein the cell is selected from HeLa, 293 T HEKand CHO cells.
 28. A method according to claim 24, wherein the cell is aprokaryotic cell.
 29. A method for down-regulation of a caspase-8, insituations where excessive cell death by apoptosis occurs, comprisingcausing said caspase-8 to come into contact with a polypeptide accordingto claim 1, wherein caspase-8 activity is inhibited.
 30. The methodaccording to claim 29, wherein said for polypeptide consists of (SEQ IDNO: 4).
 31. The method according to claim 29, wherein said polypeptideconsists of (SEQ ID NO: 5).
 32. The method according to claim 29,wherein the apoptosis is induced by the TNF receptor signaling pathway.33. A composition comprising a therapeutically effective amount of thepolypeptide according to claim
 1. 34. A composition according to claim33, wherein the polypeptide is Cari D600E mutant.
 35. A compositionaccording to claim 33, wherein the polypeptide consists of (SEQ ID NO:4).
 36. A composition according to 33, wherein the polypeptide consistsof (SEQ ID NO: 5).